Genetically modified rat comprising a humanized TRKB locus

ABSTRACT

Non-human animal genomes, non-human animal cells, and non-human animals comprising a humanized TRKB locus and methods of making and using such non-human animal genomes, non-human animal cells, and non-human animals are provided. Non-human animal cells or non-human animals comprising a humanized TRKB locus express a human TRKB protein or a chimeric transthyretin protein, fragments of which are from human TRKB. Methods are provided for using such non-human animals comprising a humanized TRKB locus to assess in vivo efficacy of human-TRKB-targeting reagents such as nuclease agents designed to target human TRKB.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 62/592,905,filed Nov. 30, 2017, and U.S. Application No. 62/661,373, filed Apr. 23,2018, each of which is herein incorporated by reference in its entiretyfor all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The Sequence Listing written in file 523129SEQLIST.txt is 154 kilobytes,was created on Nov. 30, 2018, and is hereby incorporated by reference.

BACKGROUND

Tropomyosin receptor kinase B (TRKB) is a promising target forneuroprotection in neurodegenerative diseases such as glaucoma. TRKB isone of the most widely distributed neurotrophic receptors (NTRs) in thebrain, which is highly enriched in the neocortex, hippocampus, striatum,and brainstem. Binding of brain-derived neurotrophic factor (BDNF) toTRKB receptor triggers its dimerization through conformational changesand autophosphorylation of tyrosine residues in the intracellulardomain, resulting in activation of signaling pathways involvingmitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase(PI3K), and phospholipase C-γ (PLC-γ).

TRKB is important for neuronal survival, differentiation, and function,and TRKB agonists could have therapeutic potential in numerousneurological, mental, and metabolic disorders. However, there remains aneed for suitable non-human animals providing the true human target or aclose approximation of the true human target of human-TRKB-targetingreagents, thereby enabling testing of the efficacy and mode of action ofsuch agents in live animals as well as pharmacokinetic andpharmacodynamics studies.

SUMMARY

Non-human animals comprising a humanized TRKB locus are provided, aswell as methods of using such non-human animals. Non-human animalgenomes or cells comprising a humanized TRKB locus are also provided.

In one aspect, provided are non-human animal genomes, non-human animalcells, or non-human animals comprising a humanized TRKB locus. Suchnon-human animal genomes, non-human animal cells, or non-human animalscan comprise a genetically modified endogenous TrkB locus encoding aTRKB protein, wherein the TRKB protein comprises a cytoplasmic domain, atransmembrane domain, and an extracellular domain, and all or part ofthe extracellular domain is encoded by a segment of the endogenous TrkBlocus that has been deleted and replaced with an orthologous human TRKBsequence.

In one aspect, provided are non-human animals comprising a humanizedTrkB locus. Such non-human animals can comprise a genetically modifiedendogenous TrkB locus encoding a TRKB protein, wherein the TRKB proteincomprises a cytoplasmic domain, a transmembrane domain, and anextracellular domain, and all or part of the extracellular domain isencoded by a segment of the endogenous TrkB locus that has been deletedand replaced with an orthologous human TRKB sequence.

In another aspect, provided are non-human animal cells comprising intheir genome a genetically modified endogenous TrkB locus encoding aTRKB protein, wherein the TRKB protein comprises a cytoplasmic domain, atransmembrane domain, and an extracellular domain, and all or part ofthe extracellular domain is encoded by a segment of the endogenous TrkBlocus that has been deleted and replaced with an orthologous human TRKBsequence.

In another aspect, provided are non-human animal genomes comprising agenetically modified endogenous TrkB locus encoding a TRKB protein,wherein the TRKB protein comprises a cytoplasmic domain, a transmembranedomain, and an extracellular domain, and all or part of theextracellular domain is encoded by a segment of the endogenous TrkBlocus that has been deleted and replaced with an orthologous human TRKBsequence.

In some such non-human animal genomes, non-human animal cells, ornon-human animals, the TRKB protein comprises a human TRKB extracellulardomain. Optionally, the extracellular domain comprises the sequence setforth in SEQ ID NO: 60. Optionally, all of the extracellular domain isencoded by the segment of the endogenous TrkB locus that has beendeleted and replaced with the orthologous human TRKB sequence,optionally wherein the coding sequence for the extracellular domaincomprises the sequence set forth in SEQ ID NO: 72.

In some such non-human animal genomes, non-human animal cells, ornon-human animals, the TRKB protein comprises an endogenous signalpeptide. Optionally, the signal peptide comprises the sequence set forthin SEQ ID NO: 51 or 55. Optionally, all of the signal peptide is encodedby an endogenous TrkB sequence, optionally wherein the coding sequencefor the signal peptide comprises the sequence set forth in SEQ ID NO: 63or 67.

In some such non-human animal genomes, non-human animal cells, ornon-human animals, the TRKB protein comprises an endogenous TRKBtransmembrane domain. Optionally, the transmembrane domain comprises thesequence set forth in SEQ ID NO: 53 or 57. Optionally, all of thetransmembrane domain is encoded by an endogenous TrkB sequence,optionally wherein the coding sequence for the transmembrane domaincomprises the sequence set forth in SEQ ID NO: 65 or 69.

In some such non-human animal genomes, non-human animal cells, ornon-human animals, the TRKB protein comprises an endogenous TRKBcytoplasmic domain. Optionally, the cytoplasmic domain comprises thesequence set forth in SEQ ID NO: 54 or 58. Optionally, all of thecytoplasmic domain is encoded by an endogenous TrkB sequence, optionallywherein the coding sequence for the cytoplasmic domain comprises thesequence set forth in SEQ ID NO: 66 or 70.

In some such non-human animal genomes, non-human animal cells, ornon-human animals, the TRKB protein comprises an endogenous TRKB signalpeptide, an endogenous TRKB transmembrane domain, and an endogenous TRKBcytoplasmic domain. Optionally, the signal peptide comprises thesequence set forth in SEQ ID NO: 51, the transmembrane domain comprisesthe sequence set forth in SEQ ID NO: 53, and the cytoplasmic domaincomprises the sequence set forth in SEQ ID NO: 54. Optionally, thesignal peptide comprises the sequence set forth in SEQ ID NO: 55, thetransmembrane domain comprises the sequence set forth in SEQ ID NO: 57,and the cytoplasmic domain comprises the sequence set forth in SEQ IDNO: 58. Optionally, all of the signal peptide, all of the transmembranedomain, and all of the cytoplasmic domain are encoded by an endogenousTrkB sequence. Optionally, the coding sequence for the signal peptidecomprises the sequence set forth in SEQ ID NO: 63, the coding sequencefor the transmembrane domain comprises the sequence set forth in SEQ IDNO: 65, and the coding sequence for the cytoplasmic domain comprises thesequence set forth in SEQ ID NO: 66. Optionally, the coding sequence forthe signal peptide comprises the sequence set forth in SEQ ID NO: 67,the coding sequence for the transmembrane domain comprises the sequenceset forth in SEQ ID NO: 69, and the coding sequence for the cytoplasmicdomain comprises the sequence set forth in SEQ ID NO: 70.

In some such non-human animal genomes, non-human animal cells, ornon-human animals, the TRKB protein is a chimeric non-human animal/humanTRKB protein. Optionally, the extracellular domain is a human TRKBextracellular domain, the transmembrane domain is an endogenous TRKBprotein transmembrane domain, and the cytoplasmic domain is anendogenous TRKB protein cytoplasmic domain. Optionally, the TRKB proteincomprises the sequence set forth in SEQ ID NO: 4 or 5. Optionally, thecoding sequence of the genetically modified TrkB locus encoding the TRKBprotein comprises the sequence set forth in SEQ ID NO: 12 or 13.

Some such non-human animal genomes, non-human animal cells, or non-humananimals are heterozygous for the genetically modified endogenous TrkBlocus. Some such non-human animal genomes, non-human animal cells, ornon-human animals are homozygous for the genetically modified endogenousTrkB locus.

Some such non-human animals or mammals. Optionally, the non-human animalis a rodent. Optionally, the rodent is a rat or mouse.

Some such non-human animals are rats. Optionally, the TRKB proteincomprises the sequence set forth in SEQ ID NO: 5. Optionally, the codingsequence of the genetically modified TrkB locus encoding the TRKBprotein comprises the sequence set forth in SEQ ID NO: 13.

Some such non-human animals are mice. Optionally, the TRKB proteincomprises the sequence set forth in SEQ ID NO: 4. Optionally, the codingsequence of the genetically modified TrkB locus encoding the TRKBprotein comprises the sequence set forth in SEQ ID NO: 12.

In another aspect, provided are methods of assessing the activity of ahuman-TRKB-targeting reagent in vivo using the above non-human animals.Some such methods comprise: (a) administering the human-TRKB-targetingreagent to the non-human animal; and (b) assessing the activity of thehuman-TRKB-targeting reagent in the non-human animal.

In some such methods, the assessed activity is neuroprotective activity.

In some such methods, step (a) comprises injecting thehuman-TRKB-targeting reagent to the non-human animal.

In some such methods, step (b) comprises assessing changes in one ormore or all of body weight, body composition, metabolism, and locomotionrelative to a control-non-human animal. Optionally, the assessingchanges in body composition comprises assessing lean mass and/or fatmass relative to a control non-human animal. Optionally, the assessingchanges in metabolism comprises assessing changes in food consumptionand/or water consumption.

In some such methods, step (b) comprises assessing TRKB phosphorylationand/or activation of the MAPK/ERK and PI3K/Akt pathways relative to acontrol non-human animal.

In some such methods, step (b) comprises assessing neuroprotectiveactivity. In some such methods, step (b) comprises assessingneuroprotective activity, and the non-human animal is a rat. In somesuch methods, step (b) comprises assessing retinal ganglion cellviability. Optionally, assessing retinal ganglion cell viabilitycomprises assessing retinal ganglion cell density. Optionally, retinalganglion cell density is measured in dissected retinas stained forretinal ganglion cells. Optionally, retinal ganglion cell viability isassessed in a complete optic nerve transection model after optic nerveinjury. Optionally, retinal ganglion cell viability is assessed in anoptic nerve crush model.

In some such methods, the human-TRKB-targeting reagent is anantigen-binding protein. Optionally, the antigen-binding protein is ahuman TRKB agonist antibody. In some such methods, thehuman-TRKB-targeting reagent is a small molecule. Optionally, the smallmolecule is a human TRKB agonist.

In another aspect, provided are targeting vectors for generating agenetically modified endogenous TrkB locus encoding a TRKB protein,wherein the TRKB protein comprises a cytoplasmic domain, a transmembranedomain, and an extracellular domain, and all or part of theextracellular domain is encoded by a segment of the endogenous TrkBlocus that has been deleted and replaced with an orthologous human TRKBsequence, and wherein the targeting vector comprises an insert nucleicacid comprising the orthologous human TRKB sequence flanked by a 5′homology arm targeting a 5′ target sequence at the endogenous TrkB locusand a 3′ homology arm targeting a 3′ target sequence at the endogenousTrkB locus.

In another aspect, provided are methods of making any of the non-humananimals described above. Some such methods can comprise (a) introducinginto a non-human animal pluripotent cell that is not a one-cell stageembryo: (i) an exogenous repair template comprising an insert nucleicacid flanked by a 5′ homology arm that hybridizes to a 5′ targetsequence at the endogenous TrkB locus and a 3′ homology arm thathybridizes to a 3′ target sequence at the endogenous TrkB locus, whereinthe insert nucleic acid comprises the orthologous human TRKB sequence;and (ii) a nuclease agent targeting a target sequence within theendogenous TrkB locus, wherein the genome is modified to comprise thegenetically modified endogenous TrkB locus; (b) introducing the modifiednon-human animal pluripotent cell into a host embryo; and (c) implantingthe host embryo into a surrogate mother to produce a geneticallymodified F0 generation non-human animal comprising the geneticallymodified endogenous TrkB locus. Optionally, the pluripotent cell is anembryonic stem (ES) cell. Optionally, the nuclease agent is a Cas9protein and a guide RNA that targets a guide RNA target sequence withinthe endogenous TrkB locus. Optionally, step (a) further comprisesintroducing into the non-human animal pluripotent cell a second guideRNA that targets a second guide RNA target sequence within theendogenous TrkB locus. Optionally, the exogenous repair template is alarge targeting vector that is at least 10 kb in length, or wherein theexogenous repair template is a large targeting vector in which the sumtotal of the 5′ homology arm and the 3′ homology arm is at least 10 kbin length.

Some such methods comprise: (a) introducing into a non-human animalone-cell stage embryo: (i) an exogenous repair template comprising aninsert nucleic acid flanked by a 5′ homology arm that hybridizes to a 5′target sequence at the endogenous TrkB locus and a 3′ homology arm thathybridizes to a 3′ target sequence at the endogenous TrkB locus, whereinthe insert nucleic acid comprises the orthologous human TRKB sequence;and (ii) a nuclease agent targeting a target sequence within theendogenous TrkB locus, wherein the genome is modified to comprise thegenetically modified endogenous TrkB locus; and (b) implanting themodified non-human animal one-cell stage embryo into a surrogate motherto produce a genetically modified F0 generation non-human animalcomprising the genetically modified endogenous TrkB locus. Optionally,the nuclease agent is a Cas9 protein and a guide RNA that targets aguide RNA target sequence within the endogenous TrkB locus. Optionally,step (a) further comprises introducing into the non-human one-cell stageembryo a second guide RNA that targets a second guide RNA targetsequence within the endogenous TrkB locus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (not to scale) shows a schematic of the targeting scheme forhumanization of the region of the mouse TrkB (mouse Ntrk2) locusencoding the extracellular domain of TRKB. The top portion of the figureshows the endogenous mouse TrkB (mouse Ntrk2) locus, and the bottomportion of the figure shows the large targeting vector.

FIG. 2 (not to scale) shows a schematic of the TAQMAN® assays forscreening humanization of the mouse TrkB (mouse Ntrk2) locus.Gain-of-allele (GOA) assays include 7138hU and 7138hD. Loss-of-allele(LOA) assays include 7138U and 7138D.

FIG. 3 shows western blots assessing total TRKB levels and phospho-TRKBlevels in homozygous humanized TRKB mice at 1 hour, 4 hours, and 18hours following direct hippocampal injection of TRKB agonist antibodyH4H9816P2 or isotype control antibody.

FIG. 4 (not to scale) shows a schematic of the targeting scheme forhumanization of the region of the rat TrkB (rat Ntrk2) locus encodingthe extracellular domain of TRKB. The top portion of the figure showsthe endogenous rat TrkB (rat Ntrk2) locus, and the bottom portion of thefigure shows the large targeting vector.

FIG. 5 (not to scale) shows a schematic of the TAQMAN® assays forscreening humanization of the rat TrkB (rat Ntrk2) locus and the guideRNA positions (guide target sequences set forth in SEQ ID NOS: 41-44)for targeting the rat TrkB (rat Ntrk2) locus. Gain-of-allele (GOA)assays include 7138hU and 7138hD. Loss-of-allele (LOA) assays includernoTU, rnoTM, and rnoTD. CRISPR assays designed to cover the region thatis disrupted by CRISPR/Cas9 targeting include rnoTGU and rnoTGD.Retention assays include rnoTAU2 and rnoTAD2.

FIG. 6 shows an alignment of the mouse, rat, and human TRKB (NTRK2)proteins.

FIG. 7 shows western blots of phospho-TrkB, total TrkB, phospho-Akt,total AKT, phospho-ERK, and total ERK at 15 minutes and 2 hours aftertreatment of primary cortical neurons isolated from postnatal day 1homozygous humanized TRKB mouse pups with various TrkB agonistantibodies or BDNF.

FIG. 8 shows pharmacokinetic profiles of anti-TrkB antibody in H4H9816P2in homozygous TrkB^(hu/hu) and wild type mice.

FIG. 9 shows cell survival in differentiated human neuroblastoma SH-SY5Ycells treated with different doses of TrkB agonist antibodies or BDNF.TrkB mAb1 is H4H9816P2; TrkB mAb2 is a control TrkB agonist antibodywith affinity for human TrkB, rat TrkB, and mouse TrkB. A human isotypecontrol antibody was used as a negative control. Data were normalized tothe serum-free media without antibodies.

FIG. 10 shows cell survival in primary mouse retinal ganglion cellstreated with different doses of TrkB agonist antibody or BDNF. TrkB mAb2is a control TrkB agonist antibody with affinity for human TrkB, ratTrkB, and mouse TrkB. Data were normalized to the serum-free mediawithout antibodies.

FIGS. 11A and 11B show retinal ganglion cell density in retinasdissected and stained for retinal ganglion cells in wild type rats andmice, respectively, following optic nerve transection and treatment withBDNF, TrkB agonist antibody, isotype control antibody, or vehiclecontrol. Rats were given BDNF (5 μg), TrkB agonist antibody (18 μg),isotype control antibody (18 μg), or vehicle control intravitreally at 3days and 10 days after optic nerve transection. Mice were given BDNF(2.5 μg), TrkB agonist antibody (10 μg), isotype control antibody (10μg), or vehicle control intravitreally at 3 days and 10 days after opticnerve transection. TrkB mAb2 is a control TrkB agonist antibody withaffinity for human TrkB, rat TrkB, and mouse TrkB.

FIGS. 12A and 12B show retinal ganglion cell density in retinasdissected and stained for retinal ganglion cells in wild type mice andrats, respectively, following optic nerve transection or optic nervecrush and treatment with various doses of BDNF. FIG. 12A shows BDNF doseresponse in an optic nerve crush (ONC) model in WT mice. FIG. 12B showsBDNF dose response in an optic nerve transection model in WT rat from0.13 μg to 30 μg.

FIG. 13A shows retinal ganglion cell density in retinas dissected andstained for retinal ganglion cells in homozygous, heterozygous, orwild-type TrkB rats given either TrkB agonist antibody or isotypecontrol antibody intravitreally at 3 and 10 days after optic nervetransection (****=p<0.0001; ***p<0.001; two way ANOVA). Retinas weredissected 14 days after transection. TrkB mAb1 is H4H9816P2.

FIG. 13B shows retinal ganglion cell density in non-injured eyesdissected from homozygous, heterozygous, or wild-type TrkB rats.

FIG. 13C shows the body weight of the human TRKB homozygous mice givenTrkB agonist antibody (H4H9816P2; TrkB) or isotype control antibody(REGN1945; Control).

FIG. 14 shows retinal ganglion cell density in retinas dissected andstained for retinal ganglion cells in human TRKB homozygous rats giveneither TrkB agonist antibody (hTrkB; H4H9816P2) or isotype controlantibody (REGN1945) intravitreally at 3 and 10 days after optic nervetransection. Retinas were dissected 14 days after transection.

FIGS. 15A and 15B show retinal ganglion cell density in retinasdissected and stained for retinal ganglion cells in human TRKBhomozygous rats given different TrkB agonist antibodies (H4H9816P2-L9,H4H9814P-L9, H4H9780P-L5, or a combination of all three) or isotypecontrol antibody (REGN1945) intravitreally at 3 and 10 days after opticnerve transection (** p<0.01; Kruskal-Wallis test compared to isotypecontrol antibody). Retinas were dissected 14 days after transection.FIG. 15A includes a naïve control (noninjured contralateral eye), andFIG. 15B does not.

FIG. 16 shows retinal ganglion cell density in retinas dissected andstained for retinal ganglion cells in wild type rats given differentTrkB agonist antibodies (H4H9780P and H4H9814P) or isotype controlantibody (REGN1945) intravitreally at 3 and 10 days after optic nervetransection. Retinas were dissected 14 days after transection.

FIGS. 17A and 17B show retinal ganglion cell density in retinasdissected and stained for retinal ganglion cells in human TRKBhomozygous mice given TrkB agonist antibody (H4H9780P) or isotypecontrol antibody (REGN1945) intravitreally at 3 and 10 days after opticnerve transection. Retinas were dissected 14 days after transection.FIG. 17A includes a normal control (noninjured contralateral eye), andFIG. 17B does not.

FIG. 17C shows the body weight of the human TRKB homozygous mice givenTrkB agonist antibody (H4H9780P) or isotype control antibody (REGN1945).

DEFINITIONS

The terms “protein,” “polypeptide,” and “peptide,” used interchangeablyherein, include polymeric forms of amino acids of any length, includingcoded and non-coded amino acids and chemically or biochemically modifiedor derivatized amino acids. The terms also include polymers that havebeen modified, such as polypeptides having modified peptide backbones.The term “domain” refers to any part of a protein or polypeptide havinga particular function or structure.

Proteins are said to have an “N-terminus” and a “C-terminus.” The term“N-terminus” relates to the start of a protein or polypeptide,terminated by an amino acid with a free amine group (—NH2). The term“C-terminus” relates to the end of an amino acid chain (protein orpolypeptide), terminated by a free carboxyl group (—COOH).

The terms “nucleic acid” and “polynucleotide,” used interchangeablyherein, include polymeric forms of nucleotides of any length, includingribonucleotides, deoxyribonucleotides, or analogs or modified versionsthereof. They include single-, double-, and multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purinebases, pyrimidine bases, or other natural, chemically modified,biochemically modified, non-natural, or derivatized nucleotide bases.

Nucleic acids are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. An end of an oligonucleotide is referred to as the “5′ end” ifits 5′ phosphate is not linked to the 3′ oxygen of a mononucleotidepentose ring. An end of an oligonucleotide is referred to as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of anothermononucleotide pentose ring. A nucleic acid sequence, even if internalto a larger oligonucleotide, also may be said to have 5′ and 3′ ends. Ineither a linear or circular DNA molecule, discrete elements are referredto as being “upstream” or 5′ of the “downstream” or 3′ elements.

The term “genomically integrated” refers to a nucleic acid that has beenintroduced into a cell such that the nucleotide sequence integrates intothe genome of the cell and is capable of being inherited by progenythereof. Any protocol may be used for the stable incorporation of anucleic acid into the genome of a cell.

The term “targeting vector” refers to a recombinant nucleic acid thatcan be introduced by homologous recombination,non-homologous-end-joining-mediated ligation, or any other means ofrecombination to a target position in the genome of a cell.

The term “viral vector” refers to a recombinant nucleic acid thatincludes at least one element of viral origin and includes elementssufficient for or permissive of packaging into a viral vector particle.The vector and/or particle can be utilized for the purpose oftransferring DNA, RNA, or other nucleic acids into cells either ex vivoor in vivo. Numerous forms of viral vectors are known.

The term “wild type” includes entities having a structure and/oractivity as found in a normal (as contrasted with mutant, diseased,altered, or so forth) state or context. Wild type genes and polypeptidesoften exist in multiple different forms (e.g., alleles).

The term “endogenous” refers to a nucleic acid sequence that occursnaturally within a cell or non-human animal. For example, an endogenousTrkB sequence of a non-human animal refers to a native TrkB sequencethat naturally occurs at the TrkB locus in the non-human animal.

“Exogenous” molecules or sequences include molecules or sequences thatare not normally present in a cell in that form. Normal presenceincludes presence with respect to the particular developmental stage andenvironmental conditions of the cell. An exogenous molecule or sequence,for example, can include a mutated version of a corresponding endogenoussequence within the cell, such as a humanized version of the endogenoussequence, or can include a sequence corresponding to an endogenoussequence within the cell but in a different form (i.e., not within achromosome). In contrast, endogenous molecules or sequences includemolecules or sequences that are normally present in that form in aparticular cell at a particular developmental stage under particularenvironmental conditions.

The term “heterologous” when used in the context of a nucleic acid or aprotein indicates that the nucleic acid or protein comprises at leasttwo portions that do not naturally occur together in the same molecule.For example, the term “heterologous,” when used with reference toportions of a nucleic acid or portions of a protein, indicates that thenucleic acid or protein comprises two or more sub-sequences that are notfound in the same relationship to each other (e.g., joined together) innature. As one example, a “heterologous” region of a nucleic acid vectoris a segment of nucleic acid within or attached to another nucleic acidmolecule that is not found in association with the other molecule innature. For example, a heterologous region of a nucleic acid vectorcould include a coding sequence flanked by sequences not found inassociation with the coding sequence in nature. Likewise, a“heterologous” region of a protein is a segment of amino acids within orattached to another peptide molecule that is not found in associationwith the other peptide molecule in nature (e.g., a fusion protein, or aprotein with a tag). Similarly, a nucleic acid or protein can comprise aheterologous label or a heterologous secretion or localization sequence.

“Codon optimization” takes advantage of the degeneracy of codons, asexhibited by the multiplicity of three-base pair codon combinations thatspecify an amino acid, and generally includes a process of modifying anucleic acid sequence for enhanced expression in particular host cellsby replacing at least one codon of the native sequence with a codon thatis more frequently or most frequently used in the genes of the host cellwhile maintaining the native amino acid sequence. For example, a nucleicacid encoding a Cas9 protein can be modified to substitute codons havinga higher frequency of usage in a given prokaryotic or eukaryotic cell,including a bacterial cell, a yeast cell, a human cell, a non-humancell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, ahamster cell, or any other host cell, as compared to the naturallyoccurring nucleic acid sequence. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database.” These tables canbe adapted in a number of ways. See Nakamura et al. (2000) Nucleic AcidsResearch 28:292, herein incorporated by reference in its entirety forall purposes. Computer algorithms for codon optimization of a particularsequence for expression in a particular host are also available (see,e.g., Gene Forge).

The term “locus” refers to a specific location of a gene (or significantsequence), DNA sequence, polypeptide-encoding sequence, or position on achromosome of the genome of an organism. For example, a “TrkB locus” mayrefer to the specific location of a TrkB gene, TrkB DNA sequence,TrkB-encoding sequence, or TrkB position on a chromosome of the genomeof an organism that has been identified as to where such a sequenceresides. A “TrkB locus” may comprise a regulatory element of a TrkBgene, including, for example, an enhancer, a promoter, 5′ and/or 3′untranslated region (UTR), or a combination thereof.

The term “gene” refers to a DNA sequence in a chromosome that codes fora product (e.g., an RNA product and/or a polypeptide product) andincludes the coding region interrupted with non-coding introns andsequence located adjacent to the coding region on both the 5′ and 3′ends such that the gene corresponds to the full-length mRNA (includingthe 5′ and 3′ untranslated sequences). The term “gene” also includesother non-coding sequences including regulatory sequences (e.g.,promoters, enhancers, and transcription factor binding sites),polyadenylation signals, internal ribosome entry sites, silencers,insulating sequence, and matrix attachment regions. These sequences maybe close to the coding region of the gene (e.g., within 10 kb) or atdistant sites, and they influence the level or rate of transcription andtranslation of the gene.

The term “allele” refers to a variant form of a gene. Some genes have avariety of different forms, which are located at the same position, orgenetic locus, on a chromosome. A diploid organism has two alleles ateach genetic locus. Each pair of alleles represents the genotype of aspecific genetic locus. Genotypes are described as homozygous if thereare two identical alleles at a particular locus and as heterozygous ifthe two alleles differ.

A “promoter” is a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particularpolynucleotide sequence. A promoter may additionally comprise otherregions which influence the transcription initiation rate. The promotersequences disclosed herein modulate transcription of an operably linkedpolynucleotide. A promoter can be active in one or more of the celltypes disclosed herein (e.g., a eukaryotic cell, a non-human mammaliancell, a human cell, a rodent cell, a pluripotent cell, a one-cell stageembryo, a differentiated cell, or a combination thereof). A promoter canbe, for example, a constitutively active promoter, a conditionalpromoter, an inducible promoter, a temporally restricted promoter (e.g.,a developmentally regulated promoter), or a spatially restrictedpromoter (e.g., a cell-specific or tissue-specific promoter). Examplesof promoters can be found, for example, in WO 2013/176772, hereinincorporated by reference in its entirety for all purposes.

“Operable linkage” or being “operably linked” includes juxtaposition oftwo or more components (e.g., a promoter and another sequence element)such that both components function normally and allow the possibilitythat at least one of the components can mediate a function that isexerted upon at least one of the other components. For example, apromoter can be operably linked to a coding sequence if the promotercontrols the level of transcription of the coding sequence in responseto the presence or absence of one or more transcriptional regulatoryfactors. Operable linkage can include such sequences being contiguouswith each other or acting in trans (e.g., a regulatory sequence can actat a distance to control transcription of the coding sequence).

The term “variant” refers to a nucleotide sequence differing from thesequence most prevalent in a population (e.g., by one nucleotide) or aprotein sequence different from the sequence most prevalent in apopulation (e.g., by one amino acid).

The term “fragment” when referring to a protein means a protein that isshorter or has fewer amino acids than the full-length protein. The term“fragment” when referring to a nucleic acid means a nucleic acid that isshorter or has fewer nucleotides than the full-length nucleic acid. Afragment can be, for example, an N-terminal fragment (i.e., removal of aportion of the C-terminal end of the protein), a C-terminal fragment(i.e., removal of a portion of the N-terminal end of the protein), or aninternal fragment.

“Sequence identity” or “identity” in the context of two polynucleotidesor polypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins, residue positions which are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known. Typically, this involves scoring aconservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated, e.g., as implemented in theprogram PC/GENE (Intelligenetics, Mountain View, Calif.).

“Percentage of sequence identity” includes the value determined bycomparing two optimally aligned sequences (greatest number of perfectlymatched residues) over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity. Unless otherwise specified(e.g., the shorter sequence includes a linked heterologous sequence),the comparison window is the full length of the shorter of the twosequences being compared.

Unless otherwise stated, sequence identity/similarity values include thevalue obtained using GAP Version 10 using the following parameters: %identity and % similarity for a nucleotide sequence using GAP Weight of50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; %identity and % similarity for an amino acid sequence using GAP Weight of8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or anyequivalent program thereof. “Equivalent program” includes any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The term “conservative amino acid substitution” refers to thesubstitution of an amino acid that is normally present in the sequencewith a different amino acid of similar size, charge, or polarity.Examples of conservative substitutions include the substitution of anon-polar (hydrophobic) residue such as isoleucine, valine, or leucinefor another non-polar residue. Likewise, examples of conservativesubstitutions include the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, or between glycine and serine. Additionally,the substitution of a basic residue such as lysine, arginine, orhistidine for another, or the substitution of one acidic residue such asaspartic acid or glutamic acid for another acidic residue are additionalexamples of conservative substitutions. Examples of non-conservativesubstitutions include the substitution of a non-polar (hydrophobic)amino acid residue such as isoleucine, valine, leucine, alanine, ormethionine for a polar (hydrophilic) residue such as cysteine,glutamine, glutamic acid or lysine and/or a polar residue for anon-polar residue. Typical amino acid categorizations are summarizedbelow.

Alanine Ala A Nonpolar Neutral 1.8 Arginine Arg R Polar Positive −4.5Asparagine Asn N Polar Neutral −3.5 Aspartic acid Asp D Polar Negative−3.5 Cysteine Cys C Nonpolar Neutral 2.5 Glutamic acid Glu E PolarNegative −3.5 Glutamine Gln Q Polar Neutral −3.5 Glycine Gly G NonpolarNeutral −0.4 Histidine His H Polar Positive −3.2 Isoleucine Ile INonpolar Neutral 4.5 Leucine Leu L Nonpolar Neutral 3.8 Lysine Lys KPolar Positive −3.9 Methionine Met M Nonpolar Neutral 1.9 PhenylalaninePhe F Nonpolar Neutral 2.8 Proline Pro P Nonpolar Neutral −1.6 SerineSer S Polar Neutral −0.8 Threonine Thr T Polar Neutral −0.7 TryptophanTrp W Nonpolar Neutral −0.9 Tyrosine Tyr Y Polar Neutral −1.3 Valine ValV Nonpolar Neutral 4.2

A “homologous” sequence (e.g., nucleic acid sequence) includes asequence that is either identical or substantially similar to a knownreference sequence, such that it is, for example, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the knownreference sequence. Homologous sequences can include, for example,orthologous sequence and paralogous sequences. Homologous genes, forexample, typically descend from a common ancestral DNA sequence, eitherthrough a speciation event (orthologous genes) or a genetic duplicationevent (paralogous genes). “Orthologous” genes include genes in differentspecies that evolved from a common ancestral gene by speciation.Orthologs typically retain the same function in the course of evolution.“Paralogous” genes include genes related by duplication within a genome.Paralogs can evolve new functions in the course of evolution.

The term “in vitro” includes artificial environments and to processes orreactions that occur within an artificial environment (e.g., a testtube). The term “in vivo” includes natural environments (e.g., a cell ororganism or body) and to processes or reactions that occur within anatural environment. The term “ex vivo” includes cells that have beenremoved from the body of an individual and to processes or reactionsthat occur within such cells.

The term “reporter gene” refers to a nucleic acid having a sequenceencoding a gene product (typically an enzyme) that is easily andquantifiably assayed when a construct comprising the reporter genesequence operably linked to a heterologous promoter and/or enhancerelement is introduced into cells containing (or which can be made tocontain) the factors necessary for the activation of the promoter and/orenhancer elements. Examples of reporter genes include, but are notlimited, to genes encoding beta-galactosidase (lacZ), the bacterialchloramphenicol acetyltransferase (cat) genes, firefly luciferase genes,genes encoding beta-glucuronidase (GUS), and genes encoding fluorescentproteins. A “reporter protein” refers to a protein encoded by a reportergene.

The term “fluorescent reporter protein” as used herein means a reporterprotein that is detectable based on fluorescence wherein thefluorescence may be either from the reporter protein directly, activityof the reporter protein on a fluorogenic substrate, or a protein withaffinity for binding to a fluorescent tagged compound. Examples offluorescent proteins include green fluorescent proteins (e.g., GFP,GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric AzamiGreen, CopGFP, AceGFP, and ZsGreen1), yellow fluorescent proteins (e.g.,YFP, eYFP, Citrine, Venus, YPet, PhiYFP, and ZsYellow1), bluefluorescent proteins (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv,Sapphire, and T-sapphire), cyan fluorescent proteins (e.g., CFP, eCFP,Cerulean, CyPet, AmCyanl, and Midoriishi-Cyan), red fluorescent proteins(e.g., RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1,DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2,eqFP611, mRaspberry, mStrawberry, and Jred), orange fluorescent proteins(e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange,mTangerine, and tdTomato), and any other suitable fluorescent proteinwhose presence in cells can be detected by flow cytometry methods.

The term “recombination” includes any process of exchange of geneticinformation between two polynucleotides and can occur by any mechanism.Recombination in response to double-strand breaks (DSBs) occursprincipally through two conserved DNA repair pathways: non-homologousend joining (NHEJ) and homologous recombination (HR). See Kasparek &Humphrey (2011) Seminars in Cell & Dev. Biol. 22:886-897, hereinincorporated by reference in its entirety for all purposes. Likewise,repair of a target nucleic acid mediated by an exogenous donor nucleicacid can include any process of exchange of genetic information betweenthe two polynucleotides.

NHEJ includes the repair of double-strand breaks in a nucleic acid bydirect ligation of the break ends to one another or to an exogenoussequence without the need for a homologous template. Ligation ofnon-contiguous sequences by NHEJ can often result in deletions,insertions, or translocations near the site of the double-strand break.For example, NHEJ can also result in the targeted integration of anexogenous donor nucleic acid through direct ligation of the break endswith the ends of the exogenous donor nucleic acid (i.e., NHEJ-basedcapture). Such NHEJ-mediated targeted integration can be preferred forinsertion of an exogenous donor nucleic acid when homology directedrepair (HDR) pathways are not readily usable (e.g., in non-dividingcells, primary cells, and cells which perform homology-based DNA repairpoorly). In addition, in contrast to homology-directed repair, knowledgeconcerning large regions of sequence identity flanking the cleavage siteis not needed, which can be beneficial when attempting targetedinsertion into organisms that have genomes for which there is limitedknowledge of the genomic sequence. The integration can proceed vialigation of blunt ends between the exogenous donor nucleic acid and thecleaved genomic sequence, or via ligation of sticky ends (i.e., having5′ or 3′ overhangs) using an exogenous donor nucleic acid that isflanked by overhangs that are compatible with those generated by anuclease agent in the cleaved genomic sequence. See, e.g., US2011/020722, WO 2014/033644, WO 2014/089290, and Maresca et al. (2013)Genome Res. 23(3):539-546, each of which is herein incorporated byreference in its entirety for all purposes. If blunt ends are ligated,target and/or donor resection may be needed to generation regions ofmicrohomology needed for fragment joining, which may create unwantedalterations in the target sequence.

Recombination can also occur via homology directed repair (HDR) orhomologous recombination (HR). HDR or HR includes a form of nucleic acidrepair that can require nucleotide sequence homology, uses a “donor”molecule as a template for repair of a “target” molecule (i.e., the onethat experienced the double-strand break), and leads to transfer ofgenetic information from the donor to target. Without wishing to bebound by any particular theory, such transfer can involve mismatchcorrection of heteroduplex DNA that forms between the broken target andthe donor, and/or synthesis-dependent strand annealing, in which thedonor is used to resynthesize genetic information that will become partof the target, and/or related processes. In some cases, the donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotideintegrates into the target DNA. See Wang et al. (2013) Cell 153:910-918;Mandalos et al. (2012) PLOS ONE 7:e45768:1-9; and Wang et al. (2013) NatBiotechnol. 31:530-532, each of which is herein incorporated byreference in its entirety for all purposes.

The term “antigen-binding protein” includes any protein that binds to anantigen. Examples of antigen-binding proteins include an antibody, anantigen-binding fragment of an antibody, a multispecific antibody (e.g.,a bi-specific antibody), an scFV, a bis-scFV, a diabody, a triabody, atetrabody, a V-NAR, a VHH, a VL, a F(ab), a F(ab)₂, a DVD (dual variabledomain antigen-binding protein), an SVD (single variable domainantigen-binding protein), a bispecific T-cell engager (BiTE), or aDavisbody (U.S. Pat. No. 8,586,713, herein incorporated by referenceherein in its entirety for all purposes).

As used herein, the expression “anti-TRKB antibody” includes bothmonovalent antibodies with a single specificity, as well as bispecificantibodies comprising a first arm that binds TRKB and a second arm thatbinds a second (target) antigen, wherein the anti-TRKB arm comprises,for example, any of the HCVR/LCVR or CDR sequences as set forth in Table22 herein. The expression “anti-TrkB antibody” also includesantibody-drug conjugates (ADCs) comprising an anti-TRKB antibody orantigen-binding portion thereof conjugated to a drug or toxin (i.e.,cytotoxic agent). The expression “anti-TRKB antibody” also includesantibody-radionuclide conjugates (ARCs) comprising an anti-TRKB antibodyor antigen-binding portion thereof conjugated to a radionuclide.

The term “anti-TRKB antibody” as used herein means any antigen-bindingmolecule or molecular complex comprising at least one complementaritydetermining region (CDR) that specifically binds to or interacts withTRKB or a portion of TRKB. The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-TRKB antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody” as used herein also includes antigen-bindingfragments of full length antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any enzymatically obtainable,synthetic, or genetically engineered polypeptide or glycoprotein thatspecifically binds an antigen to form a complex. Antigen-bindingfragments of an antibody may be derived, for example, from full antibodymolecules using any suitable standard techniques such as proteolyticdigestion or recombinant genetic engineering techniques involving themanipulation and expression of DNA encoding antibody variable andoptionally constant domains. Such DNA is known and/or is readilyavailable from, e.g., commercial sources, DNA libraries (including,e.g., phage-antibody libraries), or can be synthesized. The DNA may besequenced and manipulated chemically or by using molecular biologytechniques, for example, to arrange one or more variable and/or constantdomains into a suitable configuration, or to introduce codons, createcysteine residues, modify, add or delete amino acids, and so forth.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)I-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “specifically binds,” or “binds specifically to,” or the like,means that an antibody or antigen-binding fragment thereof forms acomplex with an antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by an equilibriumdissociation constant of at least about 1×10⁻⁶ M or less (e.g., asmaller K_(D) denotes a tighter binding). Methods for determiningwhether two molecules specifically bind are well-known and include, forexample, equilibrium dialysis, surface plasmon resonance, and the like.As described herein, antibodies have been identified by surface plasmonresonance, e.g., BIACORE™, which bind specifically to TRKB. Moreover,multi-specific antibodies that bind to TRKB protein and one or moreadditional antigens or a bi-specific that binds to two different regionsof TRKB are nonetheless considered antibodies that “specifically bind,”as used herein.

The anti-TRKB antibodies disclosed herein may comprise one or more aminoacid substitutions, insertions and/or deletions in the framework and/orCDR regions of the heavy and light chain variable domains. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to sequences available from, for example,public antibody sequence databases. Once obtained, antibodies andantigen-binding fragments that contain one or more mutations can beeasily tested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, and so forth. Antibodies and antigen-bindingfragments obtained in this general manner are included.

Also included are anti-TRKB antibodies comprising variants of any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein having oneor more conservative substitutions. For example, the present inventionincludes anti-TRKB antibodies having HCVR, LCVR, and/or CDR amino acidsequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer,etc. conservative amino acid substitutions relative to any of the HCVR,LCVR, and/or CDR amino acid sequences set forth in Table 22.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof in the context ofanti-TRKB antibodies, indicates that, when optimally aligned withappropriate nucleotide insertions or deletions with another nucleic acid(or its complementary strand), there is nucleotide sequence identity inat least about 95%, and more preferably at least about 96%, 97%, 98% or99% of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed below. Anucleic acid molecule having substantial identity to a reference nucleicacid molecule may, in certain instances, encode a polypeptide having thesame or substantially similar amino acid sequence as the polypeptideencoded by the reference nucleic acid molecule.

As applied to polypeptides in the context of anti-TRKB antibodies, theterm “substantial similarity” or “substantially similar” means that twopeptide sequences, when optimally aligned, such as by the programs GAPor BESTFIT using default gap weights, share at least 95% sequenceidentity, even more preferably at least 98% or 99% sequence identity.Preferably, residue positions which are not identical differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” as applied to polypeptides in the context of anti-TRKBantibodies is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in theart. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, hereinincorporated by reference in its entirety for all purposes. Examples ofgroups of amino acids that have side chains with similar chemicalproperties include: (1) aliphatic side chains: glycine, alanine, valine,leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine andthreonine; (3) amide-containing side chains: asparagine and glutamine;(4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5)basic side chains: lysine, arginine, and histidine; (6) acidic sidechains: aspartate and glutamate; and (7) sulfur-containing side chainsare cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256:1443-1445, herein incorporated by reference in its entirety for allpurposes. A “moderately conservative” replacement is any change having anonnegative value in the PAM250 log-likelihood matrix.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” a protein maycontain the protein alone or in combination with other ingredients. Thetransitional phrase “consisting essentially of” means that the scope ofa claim is to be interpreted to encompass the specified elements recitedin the claim and those that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. Thus, the term “consistingessentially of” when used in a claim of this invention is not intendedto be interpreted to be equivalent to “comprising.”

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur and that the description includesinstances in which the event or circumstance occurs and instances inwhich it does not.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (e.g., SEM) of astated value.

The term “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “or” refers to any one member of a particular list and alsoincludes any combination of members of that list.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a protein” or “at least one protein” can include a pluralityof proteins, including mixtures thereof.

Statistically significant means p≤0.05.

DETAILED DESCRIPTION

I. Overview

Disclosed herein are non-human animal genomes, non-human animal cells,and non-human animals comprising a humanized TRKB locus and methods ofusing such non-human animal cells and non-human animals. Non-humananimal cells or non-human animals comprising a humanized TRKB locusexpress a human TRKB protein or an chimeric TRKB protein comprising oneor more fragments of a human TRKB protein (e.g., all or part of thehuman TRKB extracellular domain).

A humanized TRKB allele (e.g., resulting from replacing all or part ofthe non-human animal genomic DNA one-for-one with orthologous humangenomic DNA) will provide the true human target or a close approximationof the true human target of human-TRKB-targeting reagents (e.g., agonistantibodies or agonist small molecules designed to target human TRKB),thereby enabling testing of the efficacy and mode of action of suchagents in live animals as well as pharmacokinetic and pharmacodynamicsstudies. For example, as shown in the working examples disclosed herein,intravitreal administration of human-TRKB-agonist antibodies has asignificant neuroprotective effect after optic nerve injury in humanizedTrkB rats.

II. Non-Human Animals Comprising a Humanized TRKB Locus

The non-human animal genomes, non-human animal cells, and non-humananimals disclosed herein comprise a humanized TRKB locus. Cells ornon-human animals comprising a humanized TRKB locus express a human TRKBprotein or a partially humanized, chimeric TRKB protein in which one ormore fragments of the native TRKB protein have been replaced withcorresponding fragments from human TRKB (e.g., all or part of theextracellular domain).

A. TRKB

The cells and non-human animals described herein comprise a humanizedTRKB locus. TRKB (also known as BDNF-NT-3 growth factors receptor,GP145-TrkB, Trk-B, TrkB, neurotrophic tyrosine kinase receptor type 2,TrkB tyrosine kinase, tropomyo sin-related kinase B, tropomyosinreceptor kinase B, neurotrophic receptor tyrosine kinase 2, and NTRK2)is encoded by the TRKB gene (also known as NTRK2, OBHD, TRK-B, andGP145-TRKB). TRKB is a receptor tyrosine kinase involved in thedevelopment and maturation of the central and the peripheral nervoussystems through regulation of neuron survival, proliferation, migration,differentiation, and synapse formation and plasticity. TRKB is areceptor for BDNF/brain-derived neurotrophic factor andNTF4/neurotrophin-4. Alternatively, TRKB can also bindNTF3/neurotrophin-3, which is less efficient in activating the receptorbut regulates neuron survival through TRKB. Upon ligand-binding, TRKBundergoes homodimerization, autophosphorylation, and activation. Thecanonical isoform of TRKB is expressed in the central and peripheralnervous system. In the central nervous system (CNS), expression isobserved in the cerebral cortex, hippocampus, thalamus, choroid plexus,granular layer of the cerebellum, brain stem, and spinal cord. In theperipheral nervous system, it is expressed in many cranial ganglia, theophthalmic nerve, the vestibular system, multiple facial structures, thesubmaxillary glands, and dorsal root ganglia.

Human TRKB maps to human 9q21.33 on chromosome 9 (NCBI RefSeq Gene ID4915; Assembly GRCh38.p7; location NC_000009.12 (84668368 . . .85027070)). The gene has been reported to have 23 exons. The wild typehuman TRKB protein has been assigned UniProt accession number Q16620. Atleast seven isoforms are known (Q16620-1 through Q16620-7). The sequencefor one isoform, Q16620-4 (identical to NCBI Accession No. NP_006171.2),is set forth in SEQ ID NO: 3. An mRNA (cDNA) encoding the canonicalisoform is assigned NCBI Accession No. AF410899.1 and is set forth inSEQ ID NO: 8. Another example of an mRNA (cDNA) encoding a human TRKBisoform is assigned RefSeq mRNA ID NM_006180.4. An exemplary codingsequence (CDS) is set forth in SEQ ID NO: 11. The full-length human TRKBprotein set forth in SEQ ID NO: 3 has 838 amino acids, including asignal peptide (amino acids 1-31), an extracellular domain (amino acids32-430), a transmembrane domain (amino acids 431-454), and a cytoplasmicdomain (amino acids 455-838). Delineations between these domains are asdesignated in UniProt. Reference to human TRKB includes the canonical(wild type) forms as well as all allelic forms and isoforms. Any otherforms of human TRKB have amino acids numbered for maximal alignment withthe wild type form, aligned amino acids being designated the samenumber. An example of another isoform of human TRKB is Q16620-1(identical to NCBI Accession No. NP_001018074.1), set forth in SEQ IDNO: 75. An mRNA (cDNA) encoding this isoform is assigned NCBI AccessionNo. NM_001018064.2 and is set forth in SEQ ID NO: 76. An exemplarycoding sequence (CDS) for this isoform (CCDS ID CCDS35050.1) is setforth in SEQ ID NO: 77.

Rat TrkB maps to rat 17p14 on chromosome 17 (NCBI RefSeq Gene ID 25054;Assembly Rnor_6.0; location NC_005116.4 (5934651 . . . 6245778,complement)). The gene has been reported to have 23 exons. The wild typerat TRKB protein has been assigned UniProt accession number Q63604. Atleast three isoforms are known (Q63604-1 through Q63604-3). The sequencefor the canonical isoform, Q63604-1 (identical to NCBI Accession No.NP_036863.1), is set forth in SEQ ID NO: 2. An mRNA (cDNA) encoding thecanonical isoform is assigned NCBI Accession No. NM_012731.2 and is setforth in SEQ ID NO: 7. Another example of an mRNA (cDNA) encoding a ratTRKB isoform is assigned RefSeq mRNA ID M55291. An exemplary codingsequence (CDS) is set forth in SEQ ID NO: 10. The canonical full-lengthrat TRKB protein set forth in SEQ ID NO: 2 has 821 amino acids,including a signal peptide (amino acids 1-31), an extracellular domain(amino acids 32-429), a transmembrane domain (amino acids 430-453), anda cytoplasmic domain (amino acids 454-821). Delineations between thesedomains are as designated in UniProt. Reference to rat TRKB includes thecanonical (wild type) forms as well as all allelic forms and isoforms.Any other forms of rat TRKB have amino acids numbered for maximalalignment with the wild type form, aligned amino acids being designatedthe same number.

Mouse TrkB maps to mouse 13 B1; 13 31.2 cM on chromosome 12 (NCBI RefSeqGene ID 18212; Assembly GRCm38.p4 (GCF_000001635.24); locationNC_000079.6 (58806569 . . . 59133970)). The gene has been reported tohave 23 exons. The wild type mouse TRKB protein has been assignedUniProt accession number P15209. At least four isoforms are known(P15209-1 through P15209-4). The sequence for the canonical isoform,P15209-1 (identical to NCBI Accession Nos. NP_001020245.1 andNP_001269890.1), is set forth in SEQ ID NO: 1. An exemplary mRNA (cDNA)isoform encoding the canonical isoform is assigned NCBI Accession No.NM_001025074.2 and is set forth in SEQ ID NO: 6. An exemplary codingsequence (CDS) (CODS ID CCDS26573.1) is set forth in SEQ ID NO: 9. Thecanonical full-length mouse TRKB protein set forth in SEQ ID NO: 1 has821 amino acids, including a signal peptide (amino acids 1-31), anextracellular domain (amino acids 32-429), a transmembrane domain (aminoacids 430-453), and a cytoplasmic domain (amino acids 454-821).Delineations between these domains are as designated in UniProt.Reference to mouse TRKB includes the canonical (wild type) forms as wellas all allelic forms and isoforms. Any other forms of mouse TRKB haveamino acids numbered for maximal alignment with the wild type form,aligned amino acids being designated the same number.

B. Humanized TRKB Loci

A humanized TRKB locus can be a TrkB locus in which the entire TrkB geneis replaced with the corresponding orthologous human TRKB sequence, orit can be a TrkB locus in which only a portion of the TrkB gene isreplaced with the corresponding orthologous human TRKB sequence (i.e.,humanized). Optionally, the corresponding orthologous human TRKBsequence is modified to be codon-optimized based on codon usage in thenon-human animal. Replaced (i.e., humanized) regions can include codingregions such as an exon, non-coding regions such as an intron, anuntranslated region, or a regulatory region (e.g., a promoter, anenhancer, or a transcriptional repressor-binding element), or anycombination thereof. As one example, exons corresponding to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, orall 23 exons of the human TRKB gene can be humanized. For example, exonscorresponding to exons 3-10 of the human TRKB gene can be humanized,including the segment of exon 2 (coding exon 1) from the codon encodingamino acid 33, beginning just after the signal peptide. Alternatively, aregion of TrkB encoding an epitope recognized by an anti-human-TRKBantigen-binding protein or a region targeted by human-TRKB-targetingreagent (e.g., a small molecule) can be humanized. Likewise, intronscorresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, or all 22 introns of the human TRKB gene can behumanized or can remain endogenous. For example, introns correspondingto the introns between exons 2 and 10 (i.e., introns 2-9, between codingexon 1 and exon 10) of the human TRKB gene can be humanized, optionallyincluding part of the intron following exon 10 (i.e., intron 10).Flanking untranslated regions including regulatory sequences can also behumanized or remain endogenous. For example, the 5′ untranslated region(UTR), the 3′UTR, or both the 5′ UTR and the 3′ UTR can be humanized, orthe 5′ UTR, the 3′UTR, or both the 5′ UTR and the 3′ UTR can remainendogenous. In a specific example, both the 5′ UTR and the 3′ UTR remainendogenous. Depending on the extent of replacement by orthologoussequences, regulatory sequences, such as a promoter, can be endogenousor supplied by the replacing human orthologous sequence. For example,the humanized TRKB locus can include the endogenous non-human animalTrkB promoter.

One or more or all of the regions encoding the signal peptide, thecytoplasmic domain, the transmembrane domain, or the extracellular canbe humanized, or one or more of such regions can remain endogenous.Exemplary coding sequences for a mouse TRKB signal peptide,extracellular domain, transmembrane domain, and cytoplasmic domain areset forth in SEQ ID NOS: 63-66, respectively. Exemplary coding sequencesfor a rat TRKB signal peptide, extracellular domain, transmembranedomain, and cytoplasmic domain are set forth in SEQ ID NOS: 67-70,respectively. Exemplary coding sequences for a human TRKB signalpeptide, extracellular domain, transmembrane domain, and cytoplasmicdomain are set forth in SEQ ID NOS: 71-74, respectively.

For example, all or part of the region of the TrkB locus encoding thesignal peptide can be humanized, and/or all or part of the region of theTrkB locus encoding the extracellular domain can be humanized, and/orall or part of the region of the TrkB locus encoding the transmembranedomain can be humanized, and/or all or part of the region of the TrkBlocus encoding the cytoplasmic domain can be humanized. In one example,all or part of the region of the TrkB locus encoding the extracellulardomain is humanized. Optionally, the CDS of the human TRKB extracellulardomain comprises, consists essentially of, or consists of a sequencethat is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 72 (or degenerates thereof). The TRKB protein can retain theactivity of the native TRKB (e.g., retains the ability to becomephosphorylated, retains the ability to activate downstream signalingpathways such as the PI3K/AKT and MAPK/ERK pathways, or retains theability to regulate neuron survival, proliferation, migration,differentiation, or synapse formation and plasticity or produce any ofthe phenotypes disclosed elsewhere herein). For example, the region ofthe TrkB locus encoding the extracellular domain can be humanized suchthat a chimeric TRKB protein is produced with an endogenous signalpeptide, an endogenous cytoplasmic domain, an endogenous transmembranedomain, and a humanized extracellular domain.

One or more of the regions encoding the signal peptide, the cytoplasmicdomain, the transmembrane domain, or the extracellular can remainendogenous. For example, the region encoding the signal peptide and/orthe cytoplasmic domain and/or the transmembrane domain can remainendogenous. Optionally, the CDS of the endogenous TRKB signal peptidecomprises, consists essentially of, or consists of a sequence that is atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:63 or 67 (or degenerates thereof). Optionally, the CDS of the endogenousTRKB transmembrane domain comprises, consists essentially of, orconsists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 65 or 69 (or degenerates thereof).Optionally, the CDS of the endogenous TRKB cytoplasmic domain comprises,consists essentially of, or consists of a sequence that is at least 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 66 or 70(or degenerates thereof). In each case, the TRKB protein can retain theactivity of the native TRKB.

The TRKB protein encoded by the humanized TRKB locus can comprise one ormore domains that are from a human TRKB protein and/or one or moredomains that are from an endogenous (i.e., native) TRKB protein.Exemplary amino acid sequences for a mouse TRKB signal peptide,extracellular domain, transmembrane domain, and cytoplasmic domain areset forth in SEQ ID NOS: 51-54, respectively. Exemplary amino acidsequences for a rat TRKB signal peptide, extracellular domain,transmembrane domain, and cytoplasmic domain are set forth in SEQ IDNOS: 55-58, respectively. Exemplary amino acid sequences for a humanTRKB signal peptide, extracellular domain, transmembrane domain, andcytoplasmic domain are set forth in SEQ ID NOS: 59-62, respectively.

The TRKB protein can comprise one or more or all of a human TRKB signalpeptide, a human TRKB extracellular domain, a human TRKB transmembranedomain, and a human TRKB cytoplasmic domain. As one example, the TRKBprotein can comprise a human TRKB extracellular domain.

The TRKB protein encoded by the humanized TRKB locus can also compriseone or more domains that are from the endogenous (i.e., native)non-human animal TRKB protein. As one example, the TRKB protein encodedby the humanized TRKB locus can comprise a signal peptide from theendogenous (i.e., native) non-human animal TRKB protein and/or acytoplasmic domain from the endogenous (i.e., native) non-human animalTRKB protein and/or a transmembrane domain from the endogenous (i.e.,native) non-human animal TRKB protein.

Domains in a chimeric TRKB protein that are from a human TRKB proteincan be encoded by a fully humanized sequence (i.e., the entire sequenceencoding that domain is replaced with the orthologous human TRKBsequence) or can be encoded by a partially humanized sequence (i.e.,some of the sequence encoding that domain is replaced with theorthologous human TRKB sequence, and the remaining endogenous (i.e.,native) sequence encoding that domain encodes the same amino acids asthe orthologous human TRKB sequence such that the encoded domain isidentical to that domain in the human TRKB protein). Likewise, domainsin a chimeric protein that are from the endogenous TRKB protein cay beencoded by a fully endogenous sequence (i.e., the entire sequenceencoding that domain is the endogenous TrkB sequence) or can be encodedby a partially humanized sequence (i.e., some of the sequence encodingthat domain is replaced with the orthologous human TRKB sequence, butthe orthologous human TRKB sequence encodes the same amino acids as thereplaced endogenous TrkB sequence such that the encoded domain isidentical to that domain in the endogenous TRKB protein). For examplepart of the region of the TrkB locus encoding the transmembrane domain(e.g., encoding the N-terminal region of the transmembrane domain) canbe replaced with orthologous human TRKB sequence, wherein the amino acidsequence of the region of the transmembrane domain encoded by theorthologous human TRKB sequence is identical to the correspondingendogenous amino acid sequence.

As one example, the TRKB protein encoded by the humanized TRKB locus cancomprise a human TRKB extracellular domain. Optionally, the human TRKBextracellular domain comprises, consists essentially of, or consists ofa sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 60. The TRKB protein retains the activity of thenative TRKB (e.g., retains the ability to become phosphorylated, retainsthe ability to activate downstream signaling pathways such as thePI3K/AKT and MAPK/ERK pathways, or retains the ability to regulateneuron survival, proliferation, migration, differentiation, or synapseformation and plasticity or produce any of the phenotypes disclosedelsewhere herein). As another example, the TRKB protein encoded by thehumanized TRKB locus can comprise an endogenous non-human animal TRKBcytoplasmic domain (e.g., a mouse TRKB cytoplasmic domain or a rat TRKBcytoplasmic domain). Optionally, the non-human animal TRKB cytoplasmicdomain comprises, consists essentially of, or consists of a sequencethat is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 54 or 58. As another example, the TRKB protein encoded by thehumanized TRKB locus can comprise an endogenous non-human animal TRKBtransmembrane domain (e.g., a mouse TRKB transmembrane domain or a ratTRKB transmembrane domain). Optionally, the non-human animal TRKBtransmembrane domain comprises, consists essentially of, or consists ofa sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 53 or 57. As another example, the TRKB proteinencoded by the humanized TRKB locus can comprise an endogenous non-humananimal TRKB signal peptide (e.g., a mouse TRKB signal peptide or a ratTRKB signal peptide). Optionally, the non-human animal TRKB signalpeptide comprises, consists essentially of, or consists of a sequencethat is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 51 or 55. In each case, the TRKB protein can retain theactivity of the native TRKB. For example, the TRKB protein encoded bythe humanized TRKB locus can comprise, consist essentially of, orconsist of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 4 or 5. Optionally, the TRKB CDSencoded by the humanized TRKB locus can comprise, consist essentiallyof, or consist of a sequence that is at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 12 or 13 (or degeneratesthereof). In each case, the TRKB protein can retain the activity of thenative TRKB.

Optionally, a humanized TRKB locus can comprise other elements. Examplesof such elements can include selection cassettes, reporter genes,recombinase recognition sites, or other elements. Alternatively, thehumanized TRKB locus can lack other elements (e.g., can lack a selectionmarker or selection cassette). Examples of suitable reporter genes andreporter proteins are disclosed elsewhere herein. Examples of suitableselection markers include neomycin phosphotransferase (neo_(r)),hygromycin B phosphotransferase (hyg_(r)), puromycin-N-acetyltransferase(puro_(r)), blasticidin S deaminase (bsr_(r)), xanthine/guaninephosphoribosyl transferase (gpt), and herpes simplex virus thymidinekinase (HSV-k). Examples of recombinases include Cre, Flp, and Drerecombinases. One example of a Cre recombinase gene is Crei, in whichtwo exons encoding the Cre recombinase are separated by an intron toprevent its expression in a prokaryotic cell. Such recombinases canfurther comprise a nuclear localization signal to facilitatelocalization to the nucleus (e.g., NLS-Crei). Recombinase recognitionsites include nucleotide sequences that are recognized by asite-specific recombinase and can serve as a substrate for arecombination event. Examples of recombinase recognition sites includeFRT, FRT11, FRT71, attp, att, rox, and lox sites such as loxP, lox511,lox2272, lox16, lox71, loxM2, and lox5171.

Other elements such as reporter genes or selection cassettes can beself-deleting cassettes flanked by recombinase recognition sites. See,e.g., U.S. Pat. No. 8,697,851 and US 2013/0312129, each of which isherein incorporated by reference in its entirety for all purposes. As anexample, the self-deleting cassette can comprise a Crei gene (comprisestwo exons encoding a Cre recombinase, which are separated by an intron)operably linked to a mouse Prm1 promoter and a neomycin resistance geneoperably linked to a human ubiquitin promoter. By employing the Prm1promoter, the self-deleting cassette can be deleted specifically in malegerm cells of F0 animals. The polynucleotide encoding the selectionmarker can be operably linked to a promoter active in a cell beingtargeted. Examples of promoters are described elsewhere herein. Asanother specific example, a self-deleting selection cassette cancomprise a hygromycin resistance gene coding sequence operably linked toone or more promoters (e.g., both human ubiquitin and EM7 promoters)followed by a polyadenylation signal, followed by a Crei coding sequenceoperably linked to one or more promoters (e.g., an mPrm1 promoter),followed by another polyadenylation signal, wherein the entire cassetteis flanked by loxP sites.

The humanized TRKB locus can also be a conditional allele. For example,the conditional allele can be a multifunctional allele, as described inUS 2011/0104799, herein incorporated by reference in its entirety forall purposes. For example, the conditional allele can comprise: (a) anactuating sequence in sense orientation with respect to transcription ofa target gene; (b) a drug selection cassette (DSC) in sense or antisenseorientation; (c) a nucleotide sequence of interest (NSI) in antisenseorientation; and (d) a conditional by inversion module (COIN, whichutilizes an exon-splitting intron and an invertible gene-trap-likemodule) in reverse orientation. See, e.g., US 2011/0104799. Theconditional allele can further comprise recombinable units thatrecombine upon exposure to a first recombinase to form a conditionalallele that (i) lacks the actuating sequence and the DSC; and (ii)contains the NSI in sense orientation and the COIN in antisenseorientation. See, e.g., US 2011/0104799.

One exemplary humanized TRKB locus (e.g., a humanized mouse TrkB locusor a humanized rat TrkB locus) is one in which a region in exon 2/codingexon 1 from the codon encoding amino acid 33, beginning just after thesignal peptide (or the codon corresponding to the codon encoding aminoacid 33 in mouse TrkB, rat TrkB, or human TRKB when optimally alignedwith the mouse TrkB, rat TrkB, or human TRKB CDS, respectively) throughexon 10 (or the exon corresponding to mouse TrkB, rat TrkB, or humanTRKB exon 10 when optimally aligned with the mouse TrkB, rat TrkB, orhuman TRKB CDS, respectively), optionally including a portion of intron10, is replaced with the corresponding human sequence. The replacedregion encodes the extracellular domain of TRKB. See FIGS. 1 and 4 andSEQ ID NOS: 4 and 5.

C. Non-Human Animal Genomes, Non-Human Animal Cells, and Non-HumanAnimals Comprising a Humanized TRKB Locus

Non-human animal genomes, non-human animal cells, and non-human animalscomprising a humanized TRKB locus as described elsewhere herein areprovided. The genomes, cells, or non-human animals can be male orfemale. The genomes, cells, or non-human animals can be heterozygous orhomozygous for the humanized TRKB locus. A diploid organism has twoalleles at each genetic locus. Each pair of alleles represents thegenotype of a specific genetic locus. Genotypes are described ashomozygous if there are two identical alleles at a particular locus andas heterozygous if the two alleles differ.

The non-human animal genomes or cells provided herein can be, forexample, any non-human animal genome or cell comprising a TrkB locus ora genomic locus homologous or orthologous to the human TRKB locus. Thegenomes can be from or the cells can be eukaryotic cells, which include,for example, fungal cells (e.g., yeast), plant cells, animal cells,mammalian cells, non-human mammalian cells, and human cells. The term“animal” includes any member of the animal kingdom, including, forexample, mammals, fishes, reptiles, amphibians, birds, and worms. Amammalian cell can be, for example, a non-human mammalian cell, a rodentcell, a rat cell, a mouse cell, or a hamster cell. Other non-humanmammals include, for example, non-human primates, monkeys, apes,orangutans, cats, dogs, rabbits, horses, bulls, deer, bison, livestock(e.g., bovine species such as cows, steer, and so forth; ovine speciessuch as sheep, goats, and so forth; and porcine species such as pigs andboars). Birds include, for example, chickens, turkeys, ostrich, geese,ducks, and so forth. Domesticated animals and agricultural animals arealso included. The term “non-human” excludes humans.

The cells can also be any type of undifferentiated or differentiatedstate. For example, a cell can be a totipotent cell, a pluripotent cell(e.g., a human pluripotent cell or a non-human pluripotent cell such asa mouse embryonic stem (ES) cell or a rat ES cell), or a non-pluripotentcell. Totipotent cells include undifferentiated cells that can give riseto any cell type, and pluripotent cells include undifferentiated cellsthat possess the ability to develop into more than one differentiatedcell types. Such pluripotent and/or totipotent cells can be, forexample, ES cells or ES-like cells, such as an induced pluripotent stem(iPS) cells. ES cells include embryo-derived totipotent or pluripotentcells that are capable of contributing to any tissue of the developingembryo upon introduction into an embryo. ES cells can be derived fromthe inner cell mass of a blastocyst and are capable of differentiatinginto cells of any of the three vertebrate germ layers (endoderm,ectoderm, and mesoderm).

The cells provided herein can also be germ cells (e.g., sperm oroocytes). The cells can be mitotically competent cells ormitotically-inactive cells, meiotically competent cells ormeiotically-inactive cells. Similarly, the cells can also be primarysomatic cells or cells that are not a primary somatic cell. Somaticcells include any cell that is not a gamete, germ cell, gametocyte, orundifferentiated stem cell. For example, the cells can be neurons, suchas hippocampal neurons or cortical neurons.

Suitable cells provided herein also include primary cells. Primary cellsinclude cells or cultures of cells that have been isolated directly froman organism, organ, or tissue. Primary cells include cells that areneither transformed nor immortal. They include any cell obtained from anorganism, organ, or tissue which was not previously passed in tissueculture or has been previously passed in tissue culture but is incapableof being indefinitely passed in tissue culture. Such cells can beisolated by conventional techniques and include, for example,hippocampal neurons or cortical neurons.

Other suitable cells provided herein include immortalized cells.Immortalized cells include cells from a multicellular organism thatwould normally not proliferate indefinitely but, due to mutation oralteration, have evaded normal cellular senescence and instead can keepundergoing division. Such mutations or alterations can occur naturallyor be intentionally induced. A specific example of an immortalized cellline is a neuroblastoma cell line such as N18TG2 or T48 or a cell linesuch as the NIH-3T3 cell line. Numerous types of immortalized cells arewell known. Immortalized or primary cells include cells that aretypically used for culturing or for expressing recombinant genes orproteins.

The cells provided herein also include one-cell stage embryos (i.e.,fertilized oocytes or zygotes). Such one-cell stage embryos can be fromany genetic background (e.g., BALB/c, C57BL/6, 129, or a combinationthereof for mice), can be fresh or frozen, and can be derived fromnatural breeding or in vitro fertilization.

The cells provided herein can be normal, healthy cells, or can bediseased or mutant-bearing cells.

Non-human animals comprising a humanized TRKB locus as described hereincan be made by the methods described elsewhere herein. The term “animal”includes any member of the animal kingdom, including, for example,mammals, fishes, reptiles, amphibians, birds, and worms. In a specificexample, the non-human animal is a non-human mammal. Non-human mammalsinclude, for example, non-human primates, monkeys, apes, orangutans,cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g.,mice, rats, hamsters, and guinea pigs), and livestock (e.g., bovinespecies such as cows and steer; ovine species such as sheep and goats;and porcine species such as pigs and boars). Birds include, for example,chickens, turkeys, ostrich, geese, and ducks. Domesticated animals andagricultural animals are also included. The term “non-human animal”excludes humans. Preferred non-human animals include, for example,rodents, such as mice and rats.

The non-human animals can be from any genetic background. For example,suitable mice can be from a 129 strain, a C57BL/6 strain, a mix of 129and C57BL/6, a BALB/c strain, or a Swiss Webster strain. Examples of 129strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV,129S1/Svlm), 129S2, 129S4, 129S5, 12959/SvEvH, 129S6 (129/SvEvTac),129S7, 129S8, 129T1, and 129T2. See, e.g., Festing et al. (1999)Mammalian Genome 10:836, herein incorporated by reference in itsentirety for all purposes. Examples of C57BL strains include C57BL/A,C57BL/An, C57BL/GrFa, C57BL/Ka1_wN, C57BL/6, C57BL/6J, C57BL/6ByJ,C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/O1a. Suitablemice can also be from a mix of an aforementioned 129 strain and anaforementioned C57BL/6 strain (e.g., 50% 129 and 50% C57BL/6). Likewise,suitable mice can be from a mix of aforementioned 129 strains or a mixof aforementioned BL/6 strains (e.g., the 129S6 (129/SvEvTac) strain).

Similarly, rats can be from any rat strain, including, for example, anACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, aLEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer ratstrain such as Fisher F344 or Fisher F6. Rats can also be obtained froma strain derived from a mix of two or more strains recited above. Forexample, a suitable rat can be from a DA strain or an ACI strain. TheACI rat strain is characterized as having black agouti, with white bellyand feet and an RT1^(av1) haplotype. Such strains are available from avariety of sources including Harlan Laboratories. The Dark Agouti (DA)rat strain is characterized as having an agouti coat and an RT1^(av1)haplotype. Such rats are available from a variety of sources includingCharles River and Harlan Laboratories. Some suitable rats can be from aninbred rat strain. See, e.g., US 2014/0235933, herein incorporated byreference in its entirety for all purposes.

III. Methods of Using Non-Human Animals Comprising a Humanized TRKBLocus for Assessing Efficacy of Human-TRKB-Targeting Reagents In Vivo orEx Vivo

Various methods are provided for using the non-human animals comprisinga humanized TRKB locus as described elsewhere herein for assessing oroptimizing delivery or efficacy of human-TRKB-targeting reagents (e.g.,therapeutic agonist molecules) in vivo or ex vivo. Because the non-humananimals comprise a humanized TRKB locus, the non-human animals will moreaccurately reflect the efficacy of a human-TRKB-targeting reagent.

A. Methods of Testing Efficacy of Human-TRKB-Targeting Reagents In Vivoor Ex Vivo

Various methods are provided for assessing delivery or efficacy ofhuman-TRKB-targeting reagents in vivo using non-human animals comprisinga humanized TRKB locus as described elsewhere herein. Such methods cancomprise: (a) introducing into the non-human animal ahuman-TRKB-targeting reagent; and (b) assessing the activity of thehuman-TRKB-targeting reagent.

The human-TRKB-targeting reagent can be a human-TRKB-targeting antibodyor antigen-binding protein or any other large molecule or small moleculethat targets human TRKB. Alternatively, the human-TRKB-targeting reagentcan be any biological or chemical agent that targets the human TRKBlocus (the human TRKB gene), the human TRKB mRNA, or the human TRKBprotein. Examples of human-TRKB-targeting reagents are disclosedelsewhere herein.

Such human-TRKB-targeting reagents can be administered by any deliverymethod (e.g., injection, AAV, LNP, or HDD) as disclosed in more detailelsewhere herein and by any route of administration. Means of deliveringtherapeutic molecules and routes of administration are disclosed in moredetail elsewhere herein. In particular methods, the reagents aredelivered via injection (e.g., direct hippocampal injection,subcutaneous injection, or intravitreal injection).

Methods for assessing activity of the human-TRKB-targeting reagent arewell-known and are provided elsewhere herein. In some methods, assessingactivity of the human-TRKB-targeting reagent (e.g., agonist activity orinhibitory activity) comprises assessing TRKB activity (e.g., TRKBphosphorylation, TRKB-mediated activation of downstream signalingpathways, or TRKB-induced phenotypes) as disclosed elsewhere herein.Assessment of activity can be in any cell type, any tissue type, or anyorgan type as disclosed elsewhere herein. In some methods, assessment ofactivity is in brain tissue (e.g., hippocampus or striatum) or neurons(e.g., retinal ganglion cells, hippocampal neurons, or corticalneurons).

If the TRKB-targeting reagent is a genome editing reagent (e.g., anuclease agent), such methods can comprise assessing modification of thehumanized TRKB locus. For example, the assessing can comprise sequencingthe humanized TRKB locus in one or more cells isolated from thenon-human animal (e.g., next-generation sequencing). Assessment cancomprise isolating a target organ (e.g., brain) or tissue from thenon-human animal and assessing modification of humanized TRKB locus inthe target organ or tissue. Assessment can also comprise assessingmodification of humanized TRKB locus in two or more different cell typeswithin the target organ or tissue. Similarly, assessment can compriseisolating a non-target organ or tissue (e.g., two or more non-targetorgans or tissues) from the non-human animal and assessing modificationof humanized TRKB locus in the non-target organ or tissue.

Such methods can also comprise measuring expression levels of the mRNAproduced by the humanized TRKB locus, or by measuring expression levelsof the protein encoded by the humanized TRKB locus. For example, proteinlevels can be measured in a particular cell, tissue, or organ type(e.g., brain), or secreted levels can be measured in the serum. Methodsfor assessing expression of TRKB mRNA or protein expressed from thehumanized TRKB locus are provided elsewhere herein and are well-known.

The various methods provided above for assessing activity in vivo canalso be used to assess the activity of human-TRKB-targeting reagents exvivo as described elsewhere herein.

B. Methods of Optimizing Delivery or Efficacy of Human-TRKB-TargetingReagent In Vivo or Ex Vivo

Various methods are provided for optimizing delivery ofhuman-TRKB-targeting reagents to a cell or non-human animal oroptimizing the activity or efficacy of human-TRKB-targeting reagents invivo. Such methods can comprise, for example: (a) performing the methodof testing the efficacy of a human-TRKB-targeting reagent as describedabove a first time in a first non-human animal or first cell; (b)changing a variable and performing the method a second time in a secondnon-human animal (i.e., of the same species) or a second cell with thechanged variable; and (c) comparing the activity of thehuman-TRKB-targeting reagent in step (a) with the activity of thehuman-TRKB-targeting reagent in step (b), and selecting the methodresulting in the higher efficacy or activity.

Methods of measuring delivery, efficacy, or activity ofhuman-TRKB-targeting reagents are disclosed elsewhere herein. Higherefficacy can mean different things depending on the desired effectwithin the non-human animal or cell. For example, higher efficacy canmean higher activity and/or higher specificity. Higher activity can be,for example, activity in activating TRKB or activity in inhibiting TRKB.It can refer to a higher percentage of cells being targeted within aparticular target cell type (e.g., neurons such as retinal ganglioncells) or within a particular target tissue or organ (e.g., brain).Higher specificity can refer to higher specificity with respect to TRKBas compared to off-target effects, higher specificity with respect tothe cell type targeted, or higher specificity with respect to the tissueor organ type targeted.

The variable that is changed can be any parameter. As one example, thechanged variable can be the packaging or the delivery method by whichthe human-TRKB-targeting reagent or reagents are introduced into thecell or non-human animal. Examples of delivery methods are disclosedelsewhere herein. As another example, the changed variable can be theroute of administration for introduction of the human-TRKB-targetingreagent or reagents into the cell or non-human animal. Examples ofroutes of administration are disclosed elsewhere herein.

As another example, the changed variable can be the concentration oramount of the human-TRKB-targeting reagent or reagents introduced. Asanother example, the changed variable can be the timing of introducingthe human-TRKB-targeting reagent or reagents relative to the timing ofassessing the activity or efficacy of the reagents. As another example,the changed variable can be the number of times or frequency with whichthe human-TRKB-targeting reagent or reagents are introduced. As anotherexample, the changed variable can be the human-TRKB-targeting reagent orreagents that are introduced (e.g., comparing one reagent to a differentreagent).

C. Human-TRKB-Targeting Reagents

A human-TRKB-targeting reagent can be any reagent that targets a humanTRKB protein, a human TRKB gene, or a human TRKB mRNA. Ahuman-TRKB-targeting reagent can be, for example, an agonist (i.e., amolecule that indirectly or directly activates human TRKB) or it can bean antagonist (i.e., an inhibitor or inhibitory reagent that blockshuman TRKB activity). In a specific example, the human-TRKB-targetingreagent is a TRKB agonist. Human-TRKB-targeting reagents in the methodsdisclosed herein can be known human-TRKB-targeting reagents, can beputative human-TRKB-targeting reagents (e.g., candidate reagentsdesigned to target human TRKB), or can be reagents being screened forhuman-TRKB-targeting activity.

For example, a human-TRKB-targeting reagent can be an antigen-bindingprotein (e.g., agonist antibody) targeting an epitope of a human TRKBprotein. An example of such a reagent is the TRKB agonist antibodyH4H9816P2. Other anti-TRKB antibodies are disclosed elsewhere herein. Insome cases, the anti-TRKB antibodies bind human TRKB with a K_(D) ofless than about 200 nM as measured by surface plasmon resonance at 25°C. or at 37° C. In other cases, the anti-TRKB antibodies bind human TRKBwith a K_(D) of less than about 600 pM, less than about 300 pM, lessthan about 200 pM, less than about 150 pM, less than about 100 pM, lessthan about 80 pM, less than about 50 pM, less than about 40 pM, lessthan about 30 pM, less than about 20 pM, less than about 10 pM, lessthan about 5 pM, less than about 3 pM, or less than about 1 pM. In somecases, the anti-TRKB antibodies bind human TRKB with a dissociativehalf-life (t½) of greater than about 10 minutes as measured by surfaceplasmon resonance at 25° C. or 37° C. In other cases, the anti-TRKBantibodies bind human TRKB with a t½ of greater than about 20 minutes,greater than about 50 minutes, greater than about 100 minutes, greaterthan about 120 minutes, greater than about 150 minutes, greater thanabout 300 minutes, greater than about 350 minutes, greater than about400 minutes, greater than about 450 minutes, greater than about 500minutes, greater than about 550 minutes, greater than about 600 minutes,greater than about 700 minutes, greater than about 800 minutes, greaterthan about 900 minutes, greater than about 1000 minutes, greater thanabout 1100 minutes, or greater than about 1200 minutes. As a specificexample, the anti-TRKB antibody can comprise a set of six CDRs(HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) selected from the groups set forthin Table 22 or substantially similar sequences having at least 90%, atleast 95%, at least 98%, or at least 99% sequence identity thereto.

Other human-TRKB-targeting reagents include small molecules (e.g.,agonists) targeting a human TRKB protein. Examples of small moleculeTRKB agonists include 7,8-Dihydroxyflavone (7,8-DHF), deoxygedunin,LM22A-4 (N,N′,N″-tris(2-hydroxyethyl)-1,3,5-benzenetricarboxamide), andLM22B-10(2-[[4-[[4-[Bis-(2-hydroxyethyl)-amino]-phenyl]-(4-chloro-phenyl)-methyl]-phenyl]-(2-hydroxy-ethyl)-amino]-ethanol).See, e.g., Liu et al. (2015) Translational Neurodegeneration 5:2; Massaet al. (2010) J. Clin. Invest. 120(5):1774-1785; and Yang et al. (2016)Neuropharmacology 110:343-361, each of which is herein incorporated byreference in its entirety for all purposes. An example of aTRKB-targeting reagent that is an inhibitor is K252a. See, e.g., Yang etal. (2016) Neuropharmacology 110:343-361, herein incorporated byreference in its entirety for all purposes.

Other human-TRKB-targeting reagents include peptides or peptide mimetics(e.g., agonists) targeting a human TRKB protein. Examples of peptidemimetics that serve as human TRKB agonists are disclosed, e.g., inO'Leary et al. (2003) J. Biol. Chem. 278(28):25738-25744, hereinincorporated by reference in its entirety for all purposes.

Other human-TRKB-targeting reagents can include genome editing reagentssuch as a nuclease agent (e.g., a Clustered Regularly Interspersed ShortPalindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas)nuclease, a zinc finger nuclease (ZFN), or a TranscriptionActivator-Like Effector Nuclease (TALEN)) that cleaves a recognitionsite within the human TRKB gene. Likewise, a human-TRKB-targetingreagent can be an exogenous donor nucleic acid (e.g., a targeting vectoror single-stranded oligodeoxynucleotide (ssODN)) designed to recombinewith the human TRKB gene).

Other human-TRKB-targeting reagents can include antisenseoligonucleotides (e.g., siRNA or shRNA) targeting a human TRKB mRNA.Antisense oligonucleotides (ASOs) or antisense RNAs are short syntheticstrings of nucleotides designed to prevent the expression of a targetedprotein by selectively binding to the RNA that encodes the targetedprotein and thereby preventing translation. These compounds bind to RNAwith high affinity and selectivity through well characterizedWatson-Crick base pairing (hybridization). RNA interference (RNAi) is anendogenous cellular mechanism for controlling gene expression in whichsmall interfering RNAs (siRNAs) that are bound to the RNA-inducedsilencing complex (RISC) mediate the cleavage of target messenger RNA(mRNA).

The activity of any other known or putative human-TRKB-targeting reagentcan also be assessed using the non-human animals disclosed herein.Similarly, any other molecule can be screened for human-TRKB-targetingactivity using the non-human animals disclosed herein.

D. Administering Human-TRKB-Targeting Reagents to Non-Human Animals orCells

The methods disclosed herein can comprise introducing into a non-humananimal or cell various molecules (e.g., human-TRKB-targeting reagentssuch as antibodies or small molecules), including nucleic acids,proteins, nucleic-acid-protein complexes, peptide mimetics,antigen-binding proteins, or small molecules. “Introducing” includespresenting to the cell or non-human animal the molecule (e.g., nucleicacid or protein or small molecule) in such a manner that it gains accessto the interior of the cell or to the interior of cells within thenon-human animal. The introducing can be accomplished by any means. Ifmultiple components are introduced, they can be introducedsimultaneously or sequentially in any combination. In addition, two ormore of the components can be introduced into the cell or non-humananimal by the same delivery method or different delivery methods.Similarly, two or more of the components can be introduced into anon-human animal by the same route of administration or different routesof administration.

Molecules introduced into the non-human animal or cell can be providedin compositions comprising a carrier increasing the stability of theintroduced molecules (e.g., prolonging the period under given conditionsof storage (e.g., −20° C., 4° C., or ambient temperature) for whichdegradation products remain below a threshold, such below 0.5% by weightof the starting nucleic acid or protein; or increasing the stability invivo). Non-limiting examples of such carriers include poly(lactic acid)(PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA)microspheres, liposomes, micelles, inverse micelles, lipid cochleates,and lipid microtubules.

Various methods and compositions are provided herein to allow forintroduction of a human-TRKB-targeting reagent into a cell or non-humananimal. Methods for introducing nucleic acids into various cell typesare known and include, for example, stable transfection methods,transient transfection methods, and virus-mediated methods.

Transfection protocols as well as protocols for introducing nucleic acidsequences into cells may vary. Non-limiting transfection methods includechemical-based transfection methods using liposomes; nanoparticles;calcium phosphate (Graham et al. (1973) Virology 52 (2): 456-67,Bacchetti et al. (1977) Proc. Natl. Acad. Sci. USA 74 (4): 1590-4, andKriegler, M (1991). Transfer and Expression: A Laboratory Manual. NewYork: W. H. Freeman and Company. pp. 96-97); dendrimers; or cationicpolymers such as DEAE-dextran or polyethylenimine. Non-chemical methodsinclude electroporation, Sono-poration, and optical transfection.Particle-based transfection includes the use of a gene gun, ormagnet-assisted transfection (Bertram (2006) Current PharmaceuticalBiotechnology 7, 277-28). Viral methods can also be used fortransfection.

Introduction of human-TRKB-targeting reagents into a cell can also bemediated by electroporation, by intracytoplasmic injection, by viralinfection, by adenovirus, by adeno-associated virus, by lentivirus, byretrovirus, by transfection, by lipid-mediated transfection, or bynucleofection. Nucleofection is an improved electroporation technologythat enables nucleic acid substrates to be delivered not only to thecytoplasm but also through the nuclear membrane and into the nucleus. Inaddition, use of nucleofection in the methods disclosed herein typicallyrequires much fewer cells than regular electroporation (e.g., only about2 million compared with 7 million by regular electroporation). In oneexample, nucleofection is performed using the LONZA® NUCLEOFECTOR™system.

Introduction of human-TRKB-targeting reagents into a cell (e.g., azygote) can also be accomplished by microinjection. In zygotes (i.e.,one-cell stage embryos), microinjection can be into the maternal and/orpaternal pronucleus or into the cytoplasm. If the microinjection is intoonly one pronucleus, the paternal pronucleus is preferable due to itslarger size. Microinjection of an mRNA is preferably into the cytoplasm(e.g., to deliver mRNA directly to the translation machinery), whilemicroinjection of a protein or a polynucleotide encoding a protein orencoding an RNA is preferable into the nucleus/pronucleus.Alternatively, microinjection can be carried out by injection into boththe nucleus/pronucleus and the cytoplasm: a needle can first beintroduced into the nucleus/pronucleus and a first amount can beinjected, and while removing the needle from the one-cell stage embryo asecond amount can be injected into the cytoplasm. If a protein isinjected into the cytoplasm and needs to be targeted to the nucleus, itcan comprise a nuclear localization signal to ensure delivery to thenucleus/pronucleus. Methods for carrying out microinjection are wellknown. See, e.g., Nagy et al. (Nagy A, Gertsenstein M, Vintersten K,Behringer R., 2003, Manipulating the Mouse Embryo. Cold Spring Harbor,New York: Cold Spring Harbor Laboratory Press); see also Meyer et al.(2010) Proc. Natl. Acad. Sci. USA 107:15022-15026 and Meyer et al.(2012) Proc. Natl. Acad. Sci. USA 109:9354-9359.

Other methods for introducing human-TRKB-targeting reagents into a cellor non-human animal can include, for example, vector delivery,particle-mediated delivery, exosome-mediated delivery,lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediateddelivery, or implantable-device-mediated delivery. As specific examples,a nucleic acid or protein can be introduced into a cell or non-humananimal in a carrier such as a poly(lactic acid) (PLA) microsphere, apoly(D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome, amicelle, an inverse micelle, a lipid cochleate, or a lipid microtubule.Some specific examples of delivery to a non-human animal includehydrodynamic delivery, virus-mediated delivery (e.g., adeno-associatedvirus (AAV)-mediated delivery), and lipid-nanoparticle-mediateddelivery.

Introduction of human-TRKB-targeting reagents into cells or non-humananimals can be accomplished by hydrodynamic delivery (HDD). Hydrodynamicdelivery has emerged as a method for intracellular DNA delivery in vivo.For gene delivery to parenchymal cells, only essential DNA sequencesneed to be injected via a selected blood vessel, eliminating safetyconcerns associated with current viral and synthetic vectors. Wheninjected into the bloodstream, DNA is capable of reaching cells in thedifferent tissues accessible to the blood. Hydrodynamic delivery employsthe force generated by the rapid injection of a large volume of solutioninto the incompressible blood in the circulation to overcome thephysical barriers of endothelium and cell membranes that prevent largeand membrane-impermeable compounds from entering parenchymal cells. Inaddition to the delivery of DNA, this method is useful for the efficientintracellular delivery of RNA, proteins, and other small compounds invivo. See, e.g., Bonamassa et al. (2011) Pharm. Res. 28(4):694-701,herein incorporated by reference in its entirety for all purposes.

Introduction of human-TRKB-targeting reagents can also be accomplishedby virus-mediated delivery, such as AAV-mediated delivery orlentivirus-mediated delivery. Other exemplary viruses/viral vectorsinclude retroviruses, adenoviruses, vaccinia viruses, poxviruses, andherpes simplex viruses. The viruses can infect dividing cells,non-dividing cells, or both dividing and non-dividing cells. The virusescan integrate into the host genome or alternatively do not integrateinto the host genome. Such viruses can also be engineered to havereduced immunity. The viruses can be replication-competent or can bereplication-defective (e.g., defective in one or more genes necessaryfor additional rounds of virion replication and/or packaging). Virusescan cause transient expression, long-lasting expression (e.g., at least1 week, 2 weeks, 1 month, 2 months, or 3 months), or permanentexpression. Exemplary viral titers (e.g., AAV titers) include 10¹²,10¹³, 10¹⁴, 10¹⁵, and 10¹⁶ vector genomes/mL.

The ssDNA AAV genome consists of two open reading frames, Rep and Cap,flanked by two inverted terminal repeats that allow for synthesis of thecomplementary DNA strand. When constructing an AAV transfer plasmid, thetransgene is placed between the two ITRs, and Rep and Cap can besupplied in trans. In addition to Rep and Cap, AAV can require a helperplasmid containing genes from adenovirus. These genes (E4, E2a, and VA)mediated AAV replication. For example, the transfer plasmid, Rep/Cap,and the helper plasmid can be transfected into HEK293 cells containingthe adenovirus gene E1+ to produce infectious AAV particles.Alternatively, the Rep, Cap, and adenovirus helper genes may be combinedinto a single plasmid. Similar packaging cells and methods can be usedfor other viruses, such as retroviruses.

Multiple serotypes of AAV have been identified. These serotypes differin the types of cells they infect (i.e., their tropism), allowingpreferential transduction of specific cell types. Serotypes for CNStissue include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. Serotypes forheart tissue include AAV1, AAV8, and AAV9. Serotypes for kidney tissueinclude AAV2. Serotypes for lung tissue include AAV4, AAV5, AAV6, andAAV9. Serotypes for pancreas tissue include AAV8. Serotypes forphotoreceptor cells include AAV2, AAV5, and AAV8. Serotypes for retinalpigment epithelium tissue include AAV1, AAV2, AAV4, AAV5, and AAV8.Serotypes for skeletal muscle tissue include AAV1, AAV6, AAV7, AAV8, andAAV9. Serotypes for liver tissue include AAV7, AAV8, and AAV9, andparticularly AAV8.

Tropism can be further refined through pseudotyping, which is the mixingof a capsid and a genome from different viral serotypes. For exampleAAV2/5 indicates a virus containing the genome of serotype 2 packaged inthe capsid from serotype 5. Use of pseudotyped viruses can improvetransduction efficiency, as well as alter tropism. Hybrid capsidsderived from different serotypes can also be used to alter viraltropism. For example, AAV-DJ contains a hybrid capsid from eightserotypes and displays high infectivity across a broad range of celltypes in vivo. AAV-DJ8 is another example that displays the propertiesof AAV-DJ but with enhanced brain uptake. AAV serotypes can also bemodified through mutations. Examples of mutational modifications of AAV2include Y444F, Y500F, Y730F, and S662V. Examples of mutationalmodifications of AAV3 include Y705F, Y731F, and T492V. Examples ofmutational modifications of AAV6 include S663V and T492V. Otherpseudotyped/modified AAV variants include AAV2/1, AAV2/6, AAV2/7,AAV2/8, AAV2/9, AAV2.5, AAV8.2, and AAV/SASTG.

To accelerate transgene expression, self-complementary AAV (scAAV)variants can be used. Because AAV depends on the cell's DNA replicationmachinery to synthesize the complementary strand of the AAV'ssingle-stranded DNA genome, transgene expression may be delayed. Toaddress this delay, scAAV containing complementary sequences that arecapable of spontaneously annealing upon infection can be used,eliminating the requirement for host cell DNA synthesis.

To increase packaging capacity, longer transgenes may be split betweentwo AAV transfer plasmids, the first with a 3′ splice donor and thesecond with a 5′ splice acceptor. Upon co-infection of a cell, theseviruses form concatemers, are spliced together, and the full-lengthtransgene can be expressed. Although this allows for longer transgeneexpression, expression is less efficient. Similar methods for increasingcapacity utilize homologous recombination. For example, a transgene canbe divided between two transfer plasmids but with substantial sequenceoverlap such that co-expression induces homologous recombination andexpression of the full-length transgene.

Introduction of human-TRKB-targeting reagents can also be accomplishedby lipid nanoparticle (LNP)-mediated delivery. Lipid formulations canprotect biological molecules from degradation while improving theircellular uptake. Lipid nanoparticles are particles comprising aplurality of lipid molecules physically associated with each other byintermolecular forces. These include microspheres (including unilamellarand multilamellar vesicles, e.g., liposomes), a dispersed phase in anemulsion, micelles, or an internal phase in a suspension. Such lipidnanoparticles can be used to encapsulate one or more nucleic acids orproteins for delivery. Formulations which contain cationic lipids areuseful for delivering polyanions such as nucleic acids. Other lipidsthat can be included are neutral lipids (i.e., uncharged or zwitterioniclipids), anionic lipids, helper lipids that enhance transfection, andstealth lipids that increase the length of time for which nanoparticlescan exist in vivo. Examples of suitable cationic lipids, neutral lipids,anionic lipids, helper lipids, and stealth lipids can be found in WO2016/010840 A1, herein incorporated by reference in its entirety for allpurposes. An exemplary lipid nanoparticle can comprise a cationic lipidand one or more other components. In one example, the other componentcan comprise a helper lipid such as cholesterol. In another example, theother components can comprise a helper lipid such as cholesterol and aneutral lipid such as DSPC. In another example, the other components cancomprise a helper lipid such as cholesterol, an optional neutral lipidsuch as DSPC, and a stealth lipid such as S010, S024, S027, S031, orS033.

The mode of delivery can be selected to decrease immunogenicity. Forexample, if multiple components are delivered, they may be delivered bydifferent modes (e.g., bi-modal delivery). These different modes mayconfer different pharmacodynamics or pharmacokinetic properties on thesubject delivered molecule. For example, the different modes can resultin different tissue distribution, different half-life, or differenttemporal distribution. Some modes of delivery (e.g., delivery of anucleic acid vector that persists in a cell by autonomous replication orgenomic integration) result in more persistent expression and presenceof the molecule, whereas other modes of delivery are transient and lesspersistent (e.g., delivery of an RNA or a protein).

Administration in vivo can be by any suitable route including, forexample, parenteral, intravenous, oral, subcutaneous, intra-arterial,intracranial, intrathecal, intraperitoneal, topical, intranasal, orintramuscular. Systemic modes of administration include, for example,oral and parenteral routes. Examples of parenteral routes includeintravenous, intraarterial, intraosseous, intramuscular, intradermal,subcutaneous, intranasal, and intraperitoneal routes. A specific exampleis intravenous infusion. Nasal instillation and intravitreal injectionare other specific examples. Local modes of administration include, forexample, intrathecal, intracerebroventricular, intraparenchymal (e.g.,localized intraparenchymal delivery to the striatum (e.g., into thecaudate or into the putamen), cerebral cortex, precentral gyrus,hippocampus (e.g., into the dentate gyrus or CA3 region), temporalcortex, amygdala, frontal cortex, thalamus, cerebellum, medulla,hypothalamus, tectum, tegmentum, or substantia nigra), intraocular,intraorbital, subconjuctival, intravitreal, subretinal, and transscleralroutes. Significantly smaller amounts of the components (compared withsystemic approaches) may exert an effect when administered locally (forexample, intraparenchymal or intravitreal) compared to when administeredsystemically (for example, intravenously). Local modes of administrationmay also reduce or eliminate the incidence of potentially toxic sideeffects that may occur when therapeutically effective amounts of acomponent are administered systemically. In a specific example, ahuman-TRKB-targeting reagents is administered via direct hippocampalinjection, subcutaneous injection, or intravitreal injection.

Compositions comprising human-TRKB-targeting reagents can be formulatedusing one or more physiologically and pharmaceutically acceptablecarriers, diluents, excipients or auxiliaries. The formulation candepend on the route of administration chosen. The term “pharmaceuticallyacceptable” means that the carrier, diluent, excipient, or auxiliary iscompatible with the other ingredients of the formulation and notsubstantially deleterious to the recipient thereof.

The frequency of administration and the number of dosages can be dependon the half-life of the human-TRKB-targeting reagents and the route ofadministration among other factors. The introduction ofhuman-TRKB-targeting reagents into the cell or non-human animal can beperformed one time or multiple times over a period of time. For example,the introduction can be performed at least two times over a period oftime, at least three times over a period of time, at least four timesover a period of time, at least five times over a period of time, atleast six times over a period of time, at least seven times over aperiod of time, at least eight times over a period of time, at leastnine times over a period of times, at least ten times over a period oftime, at least eleven times, at least twelve times over a period oftime, at least thirteen times over a period of time, at least fourteentimes over a period of time, at least fifteen times over a period oftime, at least sixteen times over a period of time, at least seventeentimes over a period of time, at least eighteen times over a period oftime, at least nineteen times over a period of time, or at least twentytimes over a period of time.

E. Measuring Delivery, Activity, or Efficacy of Human-TRKB-TargetingReagents In Vivo or Ex Vivo

The methods disclosed herein can further comprise detecting or measuringactivity of human-TRKB-targeting reagents. Measuring the activity ofsuch reagents (e.g., agonist activity or inhibitor activity) cancomprise measuring TRKB activity. TRKB activity can be measured by anyknown means. For example, TRKB phosphorylation can be assessed (e.g., inthe brain or neurons), activation of downstream pathways such asPI3K/AKT and MAPK/ERK by TRKB can be assessed (e.g., in the brain orneurons, such as primary cortical neurons), or cell survival can beassessed (e.g., neuron cell survival, such as retinal ganglion cellsurvival). For example, phosphorylation or activation of downstreamsignaling pathways can be assessed at 15 minutes, 30 minutes, 1 hour, 2hours, 4 hours, or 18 hours post-dosing. Increases in TRKBphosphorylation, activation of downstream signaling pathways, or cellsurvival can be indications of TRKB activation, whereas decreases can beindications of TRKB inhibition.

In non-human animals, the assessing can comprise assessing one or moreor all of body weight, body composition, metabolism, and locomotionrelative to a control-non-human animal (e.g., at 12 hours, 24 hours, 48hours, 72 hours, 96 hours, or 120 hours post-dosing). See, e.g., Lin etal. (2008) PLoS ONE 3(4):e1900; Rios et al. (2013) Trends inNeurosciences 36(2):83-90; and Zorner et al. (2003) Biol. Psychiatry54:972-982, each of which is herein incorporated by reference in itsentirety for all purposes. Assessing changes in body composition cancomprise, for example, assessing lean mass and/or fat mass. Assessingchanges in metabolism can comprise, for example, assessing changes infood consumption and/or water consumption. Decreases in body weight, fatmass, lean mass, food intake, and water intake can be indications ofTRKB activation, whereas increases can be indications of TRKBinhibition. Increases in locomotion can be indications of TRKBactivation, whereas decreases can be indications of TRKB inhibition.

The assessing can comprise assessing neuroprotective activity. As oneexample, cell survival can be assessed in non-human animals. Forexample, rodent retinal ganglion cells (RGCs) are often used to studyneurodegenerative processes associated with axonal lesion as well as toassay neuroprotective therapies. See, e.g., Nadal-Nicolás et al. (2009)Invest. Ophthalmol. Vis. Sic. 50(8):3860-3868, herein incorporated byreference in its entirety for all purposes. Retinal ganglion cellsurvival/viability can be assessed (e.g., in a complete optic nervetransection model after optic nerve injury) following treatment with ahuman-TRKB targeting reagent relative to a control non-human animal. Forexample, retinal ganglion cell survival/viability can be assessed in acomplete optic nerve transection model after optic nerve injury. See,e.g., Nadal-Nicolás et al. (2009) Invest. Ophthalmol. Vis. Sic.50(8):3860-3868, herein incorporated by reference in its entirety forall purposes. As another example, retinal ganglion cellsurvival/viability can be assessed in an optic nerve crush model. Inthis model, the crush injury to the optic nerve leads to gradual retinalganglion cells apoptosis. See., e.g., Tang et al. (2011) J. Vis. Exp.50:2685, herein incorporated by reference in its entirety for allpurposes. Retinal ganglion cell survival/viability can be assessed, forexample, by measuring retinal ganglion cell density (e.g., in retinasdissected and stained for retinal ganglion cells). Increasedsurvival/viability can be an indication of TRKB activation, whereasdecreased survival/viability can be an indication of TRKB inhibition.

If the human-TRKB-targeting reagent is a genome editing reagent, themeasuring can comprise assessing the humanized TRKB locus formodifications. Various methods can be used to identify cells having atargeted genetic modification. The screening can comprise a quantitativeassay for assessing modification of allele (MOA) of a parentalchromosome. For example, the quantitative assay can be carried out via aquantitative PCR, such as a real-time PCR (qPCR). The real-time PCR canutilize a first primer set that recognizes the target locus and a secondprimer set that recognizes a non-targeted reference locus. The primerset can comprise a fluorescent probe that recognizes the amplifiedsequence. Other examples of suitable quantitative assays includefluorescence-mediated in situ hybridization (FISH), comparative genomichybridization, isothermic DNA amplification, quantitative hybridizationto an immobilized probe(s), INVADER® Probes, TAQMAN® Molecular Beaconprobes, or ECLIPSE™ probe technology (see, e.g., US 2005/0144655, hereinincorporated by reference in its entirety for all purposes).Next-generation sequencing (NGS) can also be used for screening.Next-generation sequencing can also be referred to as “NGS” or“massively parallel sequencing” or “high throughput sequencing.” NGS canbe used as a screening tool in addition to the MOA assays to define theexact nature of the targeted genetic modification and whether it isconsistent across cell types or tissue types or organ types.

The assessing in a non-human animal can be in any cell type from anytissue or organ. For example, the assessment can be in multiple celltypes from the same tissue or organ (e.g., the brain) or in cells frommultiple locations within the tissue or organ (e.g., hippocampus andstriatum). This can provide information about which cell types within atarget tissue or organ are being targeted or which sections of a tissueor organ are being reached by the human-TRKB-targeting reagent. Asanother example, the assessment can be in multiple types of tissue or inmultiple organs. In methods in which a particular tissue, organ, or celltype is being targeted, this can provide information about howeffectively that tissue or organ is being targeted and whether there areoff-target effects in other tissues or organs.

If the reagent is designed to inactivate the humanized TRKB locus,affect expression of the humanized TRKB locus, or prevent translation ofthe humanized TRKB mRNA, the measuring can comprise assessing humanizedTRKB mRNA or protein expression. This measuring can be within the brainor particular cell types (e.g., neurons such as retinal ganglion cells).

IV. Methods of Making Non-Human Animals Comprising a Humanized TRKBLocus

Various methods are provided for making a non-human animal genome,non-human animal cell, or non-human animal comprising a humanized TRKBlocus as disclosed elsewhere herein. Any convenient method or protocolfor producing a genetically modified organism is suitable for producingsuch a genetically modified non-human animal. See, e.g., Cho et al.(2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 andGama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each ofwhich is herein incorporated by reference in its entirety for allpurposes. Such genetically modified non-human animals can be generated,for example, through gene knock-in at a targeted TrkB locus.

For example, the method of producing a non-human animal comprising ahumanized TRKB locus can comprise: (1) modifying the genome of apluripotent cell to comprise the humanized TRKB locus; (2) identifyingor selecting the genetically modified pluripotent cell comprising thehumanized TRKB locus; (3) introducing the genetically modifiedpluripotent cell into a non-human animal host embryo; and (4) implantingand gestating the host embryo in a surrogate mother. For example, themethod of producing a non-human animal comprising a humanized TRKB locuscan comprise: (1) modifying the genome of a pluripotent cell to comprisethe humanized TRKB locus; (2) identifying or selecting the geneticallymodified pluripotent cell comprising the humanized TRKB locus; (3)introducing the genetically modified pluripotent cell into a non-humananimal host embryo; and (4) gestating the host embryo in a surrogatemother. Optionally, the host embryo comprising modified pluripotent cell(e.g., a non-human ES cell) can be incubated until the blastocyst stagebefore being implanted into and gestated in the surrogate mother toproduce an F0 non-human animal. The surrogate mother can then produce anF0 generation non-human animal comprising the humanized TRKB locus.

The methods can further comprise identifying a cell or animal having amodified target genomic locus. Various methods can be used to identifycells and animals having a targeted genetic modification.

The step of modifying the genome can, for example, utilize exogenousrepair templates (e.g., targeting vectors) to modify a TrkB locus tocomprise a humanized TRKB locus disclosed herein. As one example, thetargeting vector can be for generating a humanized TRKB gene at anendogenous TrkB locus (e.g., endogenous non-human animal TrkB locus),wherein the targeting vector comprises a 5′ homology arm targeting a 5′target sequence at the endogenous TrkB locus and a 3′ homology armtargeting a 3′ target sequence at the endogenous TrkB locus. Exogenousrepair templates can also comprise nucleic acid inserts includingsegments of DNA to be integrated in the TrkB locus. Integration of anucleic acid insert in the TrkB locus can result in addition of anucleic acid sequence of interest in the TrkB locus, deletion of anucleic acid sequence of interest in the TrkB locus, or replacement of anucleic acid sequence of interest in the TrkB locus (i.e., deletion andinsertion). The homology arms can flank an insert nucleic acidcomprising human TRKB sequence to generate the humanized TRKB locus(e.g., for deleting a segment of the endogenous TrkB locus and replacingwith an orthologous human TRKB sequence).

The exogenous repair templates can be fornon-homologous-end-joining-mediated insertion or homologousrecombination. Exogenous repair templates can comprise deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), they can be single-stranded ordouble-stranded, and they can be in linear or circular form. Forexample, a repair template can be a single-stranded oligodeoxynucleotide(ssODN).

Exogenous repair templates can also comprise a heterologous sequencethat is not present at an untargeted endogenous TrkB locus. For example,an exogenous repair template can comprise a selection cassette, such asa selection cassette flanked by recombinase recognition sites.

Some exogenous repair templates comprise homology arms. If the exogenousrepair template acid also comprises a nucleic acid insert, the homologyarms can flank the nucleic acid insert. For ease of reference, thehomology arms are referred to herein as 5′ and 3′ (i.e., upstream anddownstream) homology arms. This terminology relates to the relativeposition of the homology arms to the nucleic acid insert within theexogenous repair template. The 5′ and 3′ homology arms correspond toregions within the TrkB locus, which are referred to herein as “5′target sequence” and “3′ target sequence,” respectively.

A homology arm and a target sequence “correspond” or are “corresponding”to one another when the two regions share a sufficient level of sequenceidentity to one another to act as substrates for a homologousrecombination reaction. The term “homology” includes DNA sequences thatare either identical or share sequence identity to a correspondingsequence. The sequence identity between a given target sequence and thecorresponding homology arm found in the exogenous repair template can beany degree of sequence identity that allows for homologous recombinationto occur. For example, the amount of sequence identity shared by thehomology arm of the exogenous repair template (or a fragment thereof)and the target sequence (or a fragment thereof) can be at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity, such that the sequences undergo homologous recombination.Moreover, a corresponding region of homology between the homology armand the corresponding target sequence can be of any length that issufficient to promote homologous recombination. In some targetingvectors, the intended mutation in the endogenous TrkB locus is includedin an insert nucleic acid flanked by the homology arms.

In cells other than one-cell stage embryos, the exogenous repairtemplate can be a “large targeting vector” or “LTVEC,” which includestargeting vectors that comprise homology arms that correspond to and arederived from nucleic acid sequences larger than those typically used byother approaches intended to perform homologous recombination in cells.LTVECs also include targeting vectors comprising nucleic acid insertshaving nucleic acid sequences larger than those typically used by otherapproaches intended to perform homologous recombination in cells. Forexample, LTVECs make possible the modification of large loci that cannotbe accommodated by traditional plasmid-based targeting vectors becauseof their size limitations. For example, the targeted locus can be (i.e.,the 5′ and 3′ homology arms can correspond to) a locus of the cell thatis not targetable using a conventional method or that can be targetedonly incorrectly or only with significantly low efficiency in theabsence of a nick or double-strand break induced by a nuclease agent(e.g., a Cas protein). LTVECs can be of any length and are typically atleast 10 kb in length. The sum total of the 5′ homology arm and the 3′homology arm in an LTVEC is typically at least 10 kb.

The screening step can comprise, for example, a quantitative assay forassessing modification of allele (MOA) of a parental chromosome. Forexample, the quantitative assay can be carried out via a quantitativePCR, such as a real-time PCR (qPCR). The real-time PCR can utilize afirst primer set that recognizes the target locus and a second primerset that recognizes a non-targeted reference locus. The primer set cancomprise a fluorescent probe that recognizes the amplified sequence.

Other examples of suitable quantitative assays includefluorescence-mediated in situ hybridization (FISH), comparative genomichybridization, isothermic DNA amplification, quantitative hybridizationto an immobilized probe(s), INVADER® Probes, TAQMAN® Molecular Beaconprobes, or ECLIPSE™ probe technology (see, e.g., US 2005/0144655,incorporated herein by reference in its entirety for all purposes).

An example of a suitable pluripotent cell is an embryonic stem (ES) cell(e.g., a mouse ES cell or a rat ES cell). The modified pluripotent cellcan be generated, for example, through recombination by (a) introducinginto the cell one or more exogenous donor nucleic acids (e.g., targetingvectors) comprising an insert nucleic acid flanked, for example, by 5′and 3′ homology arms corresponding to 5′ and 3′ target sites, whereinthe insert nucleic acid comprises a human TRKB sequence to generate ahumanized TRKB locus; and (b) identifying at least one cell comprisingin its genome the insert nucleic acid integrated at the endogenous TrkBlocus (i.e., identifying at least one cell comprising the humanized TRKBlocus). The modified pluripotent cell can be generated, for example,through recombination by (a) introducing into the cell one or moretargeting vectors comprising an insert nucleic acid flanked by 5′ and 3′homology arms corresponding to 5′ and 3′ target sites, wherein theinsert nucleic acid comprises a humanized TRKB locus; and (b)identifying at least one cell comprising in its genome the insertnucleic acid integrated at the target genomic locus.

Alternatively, the modified pluripotent cell can be generated by (a)introducing into the cell: (i) a nuclease agent, wherein the nucleaseagent induces a nick or double-strand break at a target site within theendogenous TrkB locus; and (ii) one or more exogenous donor nucleicacids (e.g., targeting vectors) optionally comprising an insert nucleicacid flanked by, for example, 5′ and 3′ homology arms corresponding to5′ and 3′ target sites located in sufficient proximity to the nucleasetarget site, wherein the insert nucleic acid comprises a human TRKBsequence to generate a humanized TRKB locus; and (c) identifying atleast one cell comprising in its genome the insert nucleic acidintegrated at the endogenous TrkB locus (i.e., identifying at least onecell comprising the humanized TRKB locus). Alternatively, the modifiedpluripotent cell can be generated by (a) introducing into the cell: (i)a nuclease agent, wherein the nuclease agent induces a nick ordouble-strand break at a recognition site within the target genomiclocus; and (ii) one or more targeting vectors comprising an insertnucleic acid flanked by 5′ and 3′ homology arms corresponding to 5′ and3′ target sites located in sufficient proximity to the recognition site,wherein the insert nucleic acid comprises the humanized TRKB locus; and(c) identifying at least one cell comprising a modification (e.g.,integration of the insert nucleic acid) at the target genomic locus. Anynuclease agent that induces a nick or double-strand break into a desiredrecognition site can be used. Examples of suitable nucleases include aTranscription Activator-Like Effector Nuclease (TALEN), a zinc-fingernuclease (ZFN), a meganuclease, and Clustered Regularly InterspersedShort Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems(e.g., CRISPR/Cas9 systems) or components of such systems (e.g.,CRISPR/Cas9). See, e.g., US 2013/0309670 and US 2015/0159175, each ofwhich is herein incorporated by reference in its entirety for allpurposes.

The donor cell can be introduced into a host embryo at any stage, suchas the blastocyst stage or the pre-morula stage (i.e., the 4 cell stageor the 8 cell stage). Progeny that are capable of transmitting thegenetic modification though the germline are generated. See, e.g., U.S.Pat. No. 7,294,754, herein incorporated by reference in its entirety forall purposes.

Alternatively, the method of producing the non-human animals describedelsewhere herein can comprise: (1) modifying the genome of a one-cellstage embryo to comprise the humanized TRKB locus using the methodsdescribed above for modifying pluripotent cells; (2) selecting thegenetically modified embryo; and (3) implanting and gestating thegenetically modified embryo into a surrogate mother. Alternatively, themethod of producing the non-human animals described elsewhere herein cancomprise: (1) modifying the genome of a one-cell stage embryo tocomprise the humanized TRKB locus using the methods described above formodifying pluripotent cells; (2) selecting the genetically modifiedembryo; and (3) gestating the genetically modified embryo in a surrogatemother. Progeny that are capable of transmitting the geneticmodification though the germline are generated.

Nuclear transfer techniques can also be used to generate the non-humanmammalian animals. Briefly, methods for nuclear transfer can include thesteps of: (1) enucleating an oocyte or providing an enucleated oocyte;(2) isolating or providing a donor cell or nucleus to be combined withthe enucleated oocyte; (3) inserting the cell or nucleus into theenucleated oocyte to form a reconstituted cell; (4) implanting thereconstituted cell into the womb of an animal to form an embryo; and (5)allowing the embryo to develop. In such methods, oocytes are generallyretrieved from deceased animals, although they may be isolated also fromeither oviducts and/or ovaries of live animals. Oocytes can be maturedin a variety of well-known media prior to enucleation. Enucleation ofthe oocyte can be performed in a number of well-known manners. Insertionof the donor cell or nucleus into the enucleated oocyte to form areconstituted cell can be by microinjection of a donor cell under thezona pellucida prior to fusion. Fusion may be induced by application ofa DC electrical pulse across the contact/fusion plane (electrofusion),by exposure of the cells to fusion-promoting chemicals, such aspolyethylene glycol, or by way of an inactivated virus, such as theSendai virus. A reconstituted cell can be activated by electrical and/ornon-electrical means before, during, and/or after fusion of the nucleardonor and recipient oocyte. Activation methods include electric pulses,chemically induced shock, penetration by sperm, increasing levels ofdivalent cations in the oocyte, and reducing phosphorylation of cellularproteins (as by way of kinase inhibitors) in the oocyte. The activatedreconstituted cells, or embryos, can be cultured in well-known media andthen transferred to the womb of an animal. See, e.g., US 2008/0092249,WO 1999/005266, US 2004/0177390, WO 2008/017234, and U.S. Pat. No.7,612,250, each of which is herein incorporated by reference in itsentirety for all purposes.

The various methods provided herein allow for the generation of agenetically modified non-human F0 animal wherein the cells of thegenetically modified F0 animal comprise the humanized TRKB locus. It isrecognized that depending on the method used to generate the F0 animal,the number of cells within the F0 animal that have the humanized TRKBlocus will vary. The introduction of the donor ES cells into apre-morula stage embryo from a corresponding organism (e.g., an 8-cellstage mouse embryo) via for example, the VELOCIMOUSE® method allows fora greater percentage of the cell population of the F0 animal to comprisecells having the nucleotide sequence of interest comprising the targetedgenetic modification. For example, at least 50%, 60%, 65%, 70%, 75%,85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% of the cellular contribution of the non-human F0 animalcan comprise a cell population having the targeted modification.

The cells of the genetically modified F0 animal can be heterozygous forthe humanized TRKB locus or can be homozygous for the humanized TRKBlocus.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise, if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

BRIEF DESCRIPTION OF THE SEQUENCES

The nucleotide and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three-letter code for amino acids. The nucleotidesequences follow the standard convention of beginning at the 5′ end ofthe sequence and proceeding forward (i.e., from left to right in eachline) to the 3′ end. Only one strand of each nucleotide sequence isshown, but the complementary strand is understood to be included by anyreference to the displayed strand. When a nucleotide sequence encodingan amino acid sequence is provided, it is understood that codondegenerate variants thereof that encode the same amino acid sequence arealso provided. The amino acid sequences follow the standard conventionof beginning at the amino terminus of the sequence and proceedingforward (i.e., from left to right in each line) to the carboxy terminus.

TABLE 1 Description of Sequences. SEQ ID NO Type Description 1 ProteinMouse TRKB/NTRK2 protein (P15209-1; NP_001020245.1; NP_001269890.1) 2Protein Rat TRKB/NTRK2 protein (Q63604-1; NP_036863.1) 3 Protein HumanTRKB/NTRK2 protein (Q16620-4; NP_006171.2) 4 Protein Mouse/Human HybridTRKB/NTRK2 protein 5 Protein Rat/Human Hybrid TRKB/NTRK2 protein 6 DNAMouse TrkB/Ntrk2 cDNA (NM_001025074.2) 7 DNA Rat TrkB/Ntrk2 cDNA(NM_012731.2) 8 DNA Human TRKB/NTRK2 cDNA (AF410899.1) 9 DNA MouseTrkB/Ntrk2 CDS (CCDS ID CCDS26573.1) 10 DNA Rat TrkB/Ntrk2 CDS 11 DNAHuman TRKB/NTRK2 CDS 12 DNA Mouse/Human TRKB/NTRK2 CDS 13 DNA Rat/HumanTRKB/NTRK2 CDS 14 DNA 7138 hU Fwd 15 DNA 7138 hU Probe(FAM) 16 DNA7138hU Rev 17 DNA 7138 hD Fwd 18 DNA 7138 hD Probe(Cal) 19 DNA 7138 hDRev 20 DNA 7138U Fwd 21 DNA 7138U Probe(FAM) 22 DNA 7138U Rev 23 DNA7138D Fwd 24 DNA 7138D Probe(Cal) 25 DNA 7138D Rev 26 DNA rnoTU Fwd 27DNA rnoTU Probe (FAM) 28 DNA rnoTU Rev 29 DNA rnoTD Fwd 30 DNA rnoTDProbe (Cal-Orange) 31 DNA rnoTD Rev 32 DNA rnoTM Fwd 33 DNA rnoTM Probe(FAM) 34 DNA rnoTM Rev 35 DNA rnoTAU2 Fwd 36 DNA rnoTAU2 Probe(FAM) 37DNA rnoTAU2 Rev 38 DNA rnoTAD Fwd 39 DNA rnoTAD Probe(Cal) 40 DNA rnoTADRev 41 DNA rnoGU Guide Target 42 DNA rnoGU2 Guide Target 43 DNA rnoGDGuide Target 44 DNA rnoGD2 Guide Target 45 DNA rnoTGU Fwd 46 DNA rnoTGUProbe(FAM) 47 DNA rnoTGU Rev 48 DNA rnoTGD Fwd 49 DNA rnoTGD Probe(Cal)50 DNA rnoTGD Rev 51 Protein Mouse TRKB/NTRK2 Signal Peptide 52 ProteinMouse TRKB/NTRK2 Extracellular Domain 53 Protein Mouse TRKB/NTRK2Transmembrane Domain 54 Protein Mouse TRKB/NTRK2 Cytoplasmic Domain 55Protein Rat TRKB/NTRK2 Signal Peptide 56 Protein Rat TRKB/NTRK2Extracellular Domain 57 Protein Rat TRKB/NTRK2 Transmembrane Domain 58Protein Rat TRKB/NTRK2 Cytoplasmic Domain 59 Protein Human TRKB/NTRK2Signal Peptide 60 Protein Human TRKB/NTRK2 Extracellular Domain 61Protein Human TRKB/NTRK2 Transmembrane Domain 62 Protein HumanTRKB/NTRK2 Cytoplasmic Domain 63 DNA Mouse TrkB/Ntrk2 Signal Peptide CDS64 DNA Mouse TrkB/Ntrk2 Extracellular Domain CDS 65 DNA Mouse TrkB/Ntrk2Transmembrane Domain CDS 66 DNA Mouse TrkB/Ntrk2 Cytoplasmic Domain CDS67 DNA Rat TrkB/Ntrk2 Signal Peptide CDS 68 DNA Rat TrkB/Ntrk2Extracellular Domain CDS 69 DNA Rat TrkB/Ntrk2 Transmembrane Domain CDS70 DNA Rat TrkB/Ntrk2 Cytoplasmic Domain CDS 71 DNA Human TRKB/NTRK2Signal Peptide CDS 72 DNA Human TRKB/NTRK2 Extracellular Domain CDS 73DNA Human TRKB/NTRK2 Transmembrane Domain CDS 74 DNA Human TRKB/NTRK2Cytoplasmic Domain CDS 75 Protein Human TRKB/NTRK2 protein (Q16620-1;NP_001018074.1) 76 DNA Human TRKB/NTRK2 cDNA (NM_001018064.2) 77 DNAHuman TRKB/NTRK2 CDS (CCDS ID CCDS35050.1) 78-125 DNA/ Heavy and LightChain Variable Regions and CDRs Protein of Selected Anti-TRKB Antibodiesin Table 22 and Table 23

EXAMPLES Example 1. Generation of Mice Comprising a Humanized TRKB Locus

A large targeting vector (LTVEC) comprising a 5′ homology arm comprising41.6 kb of the mouse TrkB locus and 3′ homology arm comprising 62.4 kbof the mouse TrkB locus was generated to replace a region of 65.7 kbfrom the mouse TrkB gene encoding the mouse TRKB extracellular domainwith 74.4 kb of the corresponding human sequence of TRKB. Information onmouse and human TRKB is provided in Table 2. A description of thegeneration of the large targeting vector is provided in Table 3.Generation and use of large targeting vectors (LTVECs) derived frombacterial artificial chromosome (BAC) DNA through bacterial homologousrecombination (BHR) reactions using VELOCIGENE® genetic engineeringtechnology is described, e.g., in U.S. Pat. No. 6,586,251 and Valenzuelaet al. (2003) Nat. Biotechnol. 21(6):652-659, each of which is hereinincorporated by reference in its entirety for all purposes. Generationof LTVECs through in vitro assembly methods is described, e.g., in US2015/0376628 and WO 2015/200334, each of which is herein incorporated byreference in its entirety for all purposes.

TABLE 2 Mouse and Human TRKB/NTRK2. Official NCBI Primary Genomic SymbolGene ID Source RefSeq mRNA ID UniProt ID Assembly Location Mouse Ntrk218212 MGI: 97384 NM_001025074 P15209 GRCm38/mm10 Chr 13: 58,806,569-59,133,970 (+) Human Ntrk2 4915 HGNC: 8032 AF410899 Q16620 GRCh38/hg38Chr 9: 84,669,778- 85,027,070 (+)

TABLE 3 Mouse TrkB/Ntrk2 Large Targeting Vector. Genome Build Start EndLength (bp) 5′ Mouse Arm GRCm38/mm10 Chr13: 58,767,209 Chr13: 58,808,82141,613 Human Insert GRCh38/hg38 Chr9: 84,670,730 Chr9: 84,745,139 74,4093′ Mouse Arm GRCm38/mm10 Chr13: 58,874,563 Chr13: 58,936,986 62,424

Specifically, a region starting in exon 2 (coding exon 1; from aminoacid 32, preserving signal peptide) through exon 10, including the first137 base pairs of intron 10 and all introns between exons 2 and 10(i.e., between coding exon 1 and exon 10) was deleted from the mouseTrkB locus (preserving the mouse transmembrane domain encoded by exons10 and 11). A region including exon 2/coding exon 1 (from amino acid 32,beginning after the signal peptide) through exon 10, including the first177 base pairs of intron 10 and all introns between exons 2 and 10(i.e., between coding exon 1 and exon 10) was inserted in place of thedeleted mouse region (preserving the mouse transmembrane domain encodedby exons 10 and 11).

Sequences for the mouse TRKB signal peptide, extracellular domain,transmembrane domain, and cytoplasmic domain are set forth in SEQ IDNOS: 51-54, respectively, with the corresponding coding sequence setforth in SEQ ID NOS: 63-66, respectively. Sequences for the human TRKBsignal peptide, extracellular domain, transmembrane domain, andcytoplasmic domain are set forth in SEQ ID NOS: 59-62, respectively,with the corresponding coding sequences set forth in SEQ ID NOS: 71-74,respectively. The expected encoded chimeric TRKB protein is has mouseTRKB transmembrane and intracellular domains, a mouse TRKB signalpeptide, and a human TRKB extracellular domain. See FIG. 1. An alignmentof the mouse and human TRKB proteins in FIG. 6. The mouse and humanTrkB/TRKB coding sequences are set forth in SEQ ID NOS: 9 and 11,respectively. The mouse and human TRKB protein sequences are set forthin SEQ ID NOS: 1 and 3, respectively. The sequences for the expectedchimeric mouse/human TRKB coding sequence and the expected chimericmouse/human TRKB protein are set forth in SEQ ID NOS: 12 and 4,respectively.

To generate the mutant allele, the large targeting vector was introducedinto F1H4 mouse embryonic stem cells. Following antibiotic selection,colonies were picked, expanded, and screened by TAQMAN®. See FIG. 2.Loss-of-allele assays were performed to detect loss of the endogenousmouse allele, and gain-of-allele assays were performed to detect gain ofthe humanized allele using the primers and probes set forth in Table 4.

TABLE 4 Screening Assays. Primer/ Assay Description Probe Sequence7138 hU Upstream Fwd AGGTGGGTAGGTCCTGGAAGTG (SEQ ID NO: 14) HumanProbe (FAM) AATGCTGTCCCAAGAGTGGG (SEQ ID NO: 15) Insertion RevGTCCTGCATCCCTTGTCTTTG (SEQ ID NO: 16) 7138 hD Downstream FwdATGTGGGCGTTGTGCAGTCTC (SEQ ID NO: 17) Human Probe (Cal)CGCTGCAGTGCATTGAACTCAGCA (SEQ ID NO: 18) Insertion RevCTGTGGAGGGACGTGACCAG (SEQ ID NO: 19) 7138U Upstream FwdTCCGCTAGGATTTGGTGTACTG (SEQ ID NO: 20) Mouse LOA Probe (FAM)AGCCTTCTCCAGGCATCGTGGCAT (SEQ ID NO: 21) RevTCCGGGTCAACGCTGTTAG (SEQ ID NO: 22) 7138D Downstream FwdTCCTGCGAGGGTTCTGAC (SEQ ID NO: 23) Mouse LOA Probe (Ca)TGGGTGCTCATATGCCAGAGAAATTGTCA (SEQ ID NO: 24) RevCGATCTGTGATGGCCTGCTTAC (SEQ ID NO: 25)

Modification-of-allele (MOA) assays including loss-of-allele (LOA) andgain-of-allele (GOA) assays are described, for example, in US2014/0178879; US 2016/0145646; WO 2016/081923; and Frendewey et al.(2010) Methods Enzymol. 476:295-307, each of which is hereinincorporated by reference in its entirety for all purposes. Theloss-of-allele (LOA) assay inverts the conventional screening logic andquantifies the number of copies in a genomic DNA sample of the nativelocus to which the mutation was directed. In a correctly targetedheterozygous cell clone, the LOA assay detects one of the two nativealleles (for genes not on the X or Y chromosome), the other allele beingdisrupted by the targeted modification. The same principle can beapplied in reverse as a gain-of-allele (GOA) assay to quantify the copynumber of the inserted targeting vector in a genomic DNA sample.

F0 mice were generated using the VELOCIMOUSE® method. See, e.g., U.S.Pat. Nos. 7,576,259; 7,659,442; 7,294,754; US 2008/0078000; andPoueymirou et al. (2007) Nat. Biotechnol. 25(1):91-99, each of which isherein incorporated by reference in its entirety for all purposes. Inthe VELOCIMOUSE® method, targeted mouse embryonic stem (ES) cells areinjected through laser-assisted injection into pre-morula stage embryos,e.g., eight-cell-stage embryos, which efficiently yields F0 generationmice that are fully ES-cell-derived. All experiments performed inhumanized TRKB mice as described below were performed in mice in whichthe self-deleting selection cassette was self-deleted.

Example 2. In Vivo Comparison of Effect of H4H9816 and Isotype ControlREGN1945 Antibodies on Body Weight and Metabolism in TrkB^(hu/hu) Mice(MAID7139)

Experimental Procedure

To determine the effect of a TRKB agonist antibody, H4H9816P2, on bodyweight and composition, a metabolic study of mice homozygous for theexpression of human TRKB receptor in place of the mouse TRKB receptor(TrkB^(hu/hu) mice) was conducted following a single sub-cutaneousantibody injection. These studies were undertaken in part based onprevious studies of TrkB agonists and TrkB-knockout mice. See, e.g., Linet al. (2008) PLoS ONE 3(4):e1900; Rios et al. (2013) Trends inNeurosciences 36(2):83-90; and Zorner et al. (2003) Biol. Psychiatry54:972-982, each of which is herein incorporated by reference in itsentirety for all purposes. TrkB^(hu/hu) mice (male, 20 weeks old) werefirst transferred from group-cage to single-cage housing for two weeksof acclimatization. After this period, mice were transferred tometabolic cages (CLAMS, Columbus Instruments) to assess changes in foodand water consumption, locomotion, energy expenditure, and respirationfollowing antibody administration. Regular powdered chow was stored in afloor chamber on a spring-loaded scale (Mettler Toledo, PL602E) tomeasure food consumption via changes in total chow weight. Water wasaccessible via a cage-top spout and intake was measured by trackingchanges in pump-line volume (Oxymax®/CLAMS Liquid Unit). CLAMS metaboliccages measured each of these parameters in continuous, 16-18 minuteintervals throughout the duration of the study. Metabolic data wereanalyzed in single measures and summarized in 24-hour intervalscontaining one complete dark and light cycle using OXYMAX®/CLAMSsoftware (Columbus instruments, v5.35). After acclimating to the cagesfor two weeks, TrkB^(hu/hu) mice received a single 50 mg/kgsub-cutaneous dose of either a TRKB agonist antibody, H4H9816P2, or anIgG4 isotype control antibody in PBS at pH7.2. A group of naïve controlTrkB^(hu/hu) mice did not receive an injection. Mice were weighedimmediately prior to dosing, and at 24, 48, 72, 96, and 120 hourspost-dosing. In order to measure each mouse's body composition, NuclearMagnetic Resonance Relaxometry, also referred to as QuantitativeMagnetic Resonance, was performed using an EchoMRI™-500 Analyzer(EchoMRI LLC). Prior to dosing, mice were placed in a clear plasticholder and inserted into the NMR-MRI device to measure each subject'slean mass, fat mass, and hydration status. Measurements were performedover the course of 0.5-3.2 minutes per mouse, and were taken againapproximately 120 hours after dosing.

Results and Conclusions

Daily body weight monitoring was performed to determine whether a singlesubcutaneous injection of H4H9816P2 induces weight loss in TrkB^(hu/hu)mice. Prior to dosing, there were no significant differences in theaverage body weight of the three treatment groups, as each had anaverage pre-dose body weight of 28.39-29.85 g (Table 5). At 48 hourspost-dosing, however, H4H9816P2-treated TrkB^(hu/hu) mice lost anaverage of 1.70 g, or 5.96% of their pre-dose body weight. At the sametime point, naïve and isotype control antibody-treated TrkB^(hu/hu) micegained between 1.79-2.37% of their pre-dose body weight.H4H9816P2-treated TrkB^(hu/hu) mice continued to lose weight throughoutthe full time course of the study, and by 72 and 96 hours post-dosingthese mice had lost an average of 8.42% and 11.80% of their pre-dosebody weight, respectively. At 120 hours post-dosing, H4H9816P2-treatedTrkB^(hu/hu) mice had lost an average of 12.67% of their pre-dose bodyweight. Conversely, naïve and isotype control-treated TrkB^(hu/hu) micedid not exhibit any loss in pre-dose body weight throughout the study.As body weight in H4H9816P2-treated TrkB^(hu/hu) mice was significantlyreduced relative to both naïve and isotype controls at 48, 72, 96, and120 hours post-dosing, it was determined that TRKB agonist antibodyH4H9816P2 induced significant body weight loss in TrkB^(hu/hu) mice.

TABLE 5 Body Weight of TrkB^(hu/hu) Mice after Dosing with TRKB AgonistAntibody H4H9816P2. Mean body Mean body Mean body Mean body Mean bodyMean pre- weight (g) weight (g) weight (g) weight (g) weight (g) dosebody 24 hours 48 hours 72 hours 96 hours 120 hours weight (g) post-dosepost-dose post-dose post-dose post-dose (±SD) (±SD) (±SD) (±SD) (±SD)(±SD) Percent Percent Percent Percent Percent Percent change from changefrom change from change from change from change from pre-dose pre-dosepre-dose pre-dose pre-dose pre-dose Experimental body weight body weightbody weight body weight body weight body weight group (+/−SD) (+/−SD)(+/−SD) (+/−SD) (+/−SD) (+/−SD) Naive 28.85 29.69 29.36 29.32 29.2928.88 (n = 3) (+/−0.81) (+/−0.97) (+/−1.10) (+/−1.29) (+/−1.10)(+/−1.04) N/A +2.91%  +1.79% +1.65% +1.54% +0.10% (+/−0.62) (+/−1.62)(+/−2.24) (+/−1.22) (+/−1.05) Isotype control 29.21 30.27 29.90 30.0829.87 29.69 (n = 4) (+/−2.68) (+/−2.51) (+/−2.63) (+/−2.69) (+/−2.52)(+/−2.68) N/A +3.61% +2.37% +2.98% +2.25% +1.65% (+/−1.68) (+/−1.50)(+/−1.09) (+/−1.56) (+/−0.81) H4H9816P2 28.39 27.87 26.69 26.00* 25.04**24.79** (n = 4) (+/−1.35) (+/−1.29) (+/−0.87) (+/−0.98) (+/−1.03)(+/−1.36) N/A −1.83% −5.96% −8.42% −11.80% −12.67% (+/−0.56) (+/−1.88)(+/−1.85) (+/−1.52) (+/−1.66) Statistical significance determined bytwo-way ANOVA with Tukey's multiple comparison post-hoc test isindicated (*= p < 0.05, **= p < 0.01, *** = p < 0.001, **** = p <0.0001, compared to isotype control group: TrkB^(hu/hu) mice dosed with50 mg/kg isotype control antibody.

The effect of TRKB agonist antibody H4H9816P2 injection on bodycomposition was also measured by performing NMR-MRI on each subjectbefore and after dosing. Prior to dosing, the three treatment groups ofTrkB^(hu/hu) mice did not exhibit any significant differences in fatmass or lean mass, as each group had an average of 4.19-4.75 g of fatmass and 21.32-21.70 g of lean mass (Table 6). Following antibodyadministration, however, TrkB^(hu/hu) mice dosed with H4H9816P2 lost anaverage of 48.90% of their total body fat mass over the course of thestudy (Table 6). Naïve and isotype control antibody-treated TrkB^(hu/hu)mice lost an average of 8.49% and 9.48% of their pre-dose fat mass,respectively, which was significantly less than H4H9816P2-treatedsubjects (Table 6). Furthermore, H4H9816P2-treated TrkB^(hu/hu) micelost an average of 7.84% of their lean mass throughout the study, whichwas significantly greater than the 2.41% and 1.75% of average pre-doselean mass lost by naïve and isotype control antibody-treated groups,respectively (Table 6). As such, the described body weight loss could beexplained by a significant loss of fat mass and a modest loss of leanmass following injection of TRKB agonist antibody H4H9816P2 inTrkB^(hu/hu) mice.

TABLE 6 Body Composition of TrkB^(hu/hu) Mice after Dosing with TRKBAgonist Antibody H4H9816P2. Mean fat Mean fat Mean lean Mean lean Meanpre- mass (%) mass change Mean pre- mass (%) mass change dose fat 120hours (%) 120 hours dose lean 120 hours (%) 120 hours Experimental mass(%) post-dose post-dose mass (%) post-dose post-dose group (±SD) (±SD)(±SD) (±SD) (±SD) (±SD) Naive 4.65 4.27 −8.49 21.45 20.94 −2.41 (n = 3)(+/−0.32) (+/−0.55) (+/−7.18) (+/−0.79) (+/−0.98) (+/−1.81) Isotypecontrol 4.75 4.40 −9.48 21.70 21.32 −1.75 (n = 4) (+/−2.98) (+/−2.98)(+/−6.00) (+/−0.50) (+/−0.35) (+/−0.98) H4H9816P2 4.19 2.14   −48.90****21.32 19.64 −7.84*** (n = 4) (+/−1.15) (+/−0.64) (+/−5.06) (+/−1.87)(+/−1.69) (+/−0.94) Statistical significance determined byKruskal-Wallis One-way ANOVA with Tukey's multiple comparison post-hoctest is indicated (* = p < 0.05, ** = p < 0.01, ***= p < 0.001, ****= p< 0.0001, compared to isotype control group: TrkB^(hu/hu) mice dosedwith 50 mg/kg isotype control antibody.

In addition to assessing the effects of TRKB agonist antibody H4H9816P2injection on body weight and composition in TrkB^(hu/hu) mice, feeding,drinking, and locomotor activity were continuously measured by metaboliccages. Prior to dosing, TrkB^(hu/hu) mice consumed an average of3.49-3.73 g of chow per day. Within 24 hours of dosing, however,H4H9816P2-treated TrkB^(hu/hu) mice significantly reduced their foodintake to 2.20 g of chow per day. The average level of food intake inH4H9816P2-treated TrkB^(hu/hu) mice did not exceed 2.49 g of chow perday throughout the remainder of the study, while naïve and isotypeantibody-treated TrkB^(hu/hu) mice consistently consumed an average of3.62-4.07 g of chow per day (Table 7).

Similarly, there were no significant differences in daily waterconsumption between treatment groups prior to dosing. TrkB^(hu/hu) miceconsumed an average of 4.67-5.55 mL of water per day in each treatmentgroup (Table 8). After dosing, H4H9816P2-treated TrkB^(hu/hu) micereduced their water intake to 2.05-3.24 mL of water per day. This wassignificantly lower than naïve and isotype control antibody-treatedTrkB^(hu/hu) mice, which consistently consumed 4.50-5.77 mL of water perday throughout the study (Table 8). Thus, injection of the TRKB agonistantibody, H4H9816P2, appeared to result in a significant reduction ofboth food and water intake in TrkB^(hu/hu) mice relative to both naïveand isotype controls.

TABLE 7 Food Consumption of TrkB^(hu/hu) Mice after Dosing with TRKBAgonist Antibody H4H9816P2. Mean total Mean total Mean total Mean totalMean total food intake food intake food intake food intake food intake(g) 0-24 hours (g) 0-24 hours (g) 24-48 hours (g) 48-72 hours (g) 72-96hours pre-dose post-dose post-dose post-dose post-dose Experimentalgroup (±SD) (±SD) (±SD) (±SD) (±SD) Naive (n = 3) 3.51 3.98 3.76 3.623.91 (+/−0.53) (+/−0.08) (+/−0.19) (+/−0.35) (+/−0.18) Isotype control(n = 4) 3.73 4.07 3.99 3.89 3.80 (+/−0.48) (+/−0.23) (+/−0.17) (+/−0.22)(+/−0.22) H4H9816P2 (n = 4) 3.49   2.20****   2.08****   2.18****  2.49*** (+/−1.07) (+/−0.16) (+/−0.36) (+/−0.37) (+/−0.47) Statisticalsignificance determined by Kruskal-Wallis One-way ANOVA with Tukey'smultiple comparison post-hoc test is indicated (*= p < 0.05, **= p <0.01, ***= p < 0.001, ****= p < 0.0001, compared to isotype controlgroup: TrkB^(hu/hu) mice dosed with 50 mg/kg isotype control antibody.

TABLE 8 Water Consumption of TrkB^(hu/hu) Mice after Dosing with TrkBAgonist Antibody H4H9816P2. Mean total Mean total Mean total Mean totalMean total water intake water intake water intake water intake waterintake (mL) 0-24 (mL) 0-24 (mL) 24-48 (mL) 48-72 (mL) 72-96 hours pre-hours post- hours post- hours post- hours post- Experimental group dose(±SD) dose (±SD) dose (±SD) dose (±SD) dose (±SD) Naive (n = 3) 4.795.42 4.96 4.57 4.88 (+/−0.21) (+/−0.94) (+/−0.91) (+/−0.56) (+/−0.32)Isotype control (n = 4) 5.55 4.50 5.08 5.09 5.77 (+/−1.23) (+/−1.08)(+/−1.39) (+/−1.10) (+/−1.62) H4H9816P2 (n = 4) 4.67  2.25**  3.24*  2.05***   2.25**** (+/−1.13) (+/−0.55) (+/−1.10) (+/−0.29) (+/−0.24)Statistical significance determined by Kruskal-Wallis One-way ANOVA withTukey's multiple comparison post-hoc test is indicated (*= p < 0.05, **=p < 0.01, ***= p < 0.001, ****= p < 0.0001, compared to isotype controlgroup: TrkB^(hu/hu) mice dosed with 50 mg/kg isotype control antibody.

To determine the effects of antibody treatment on activity, locomotionwas analyzed by OXYMAX®/CLAMS software (Columbus instruments, v5.35),which continuously measured the total number of x-plane ambulations ofeach mouse. One mouse exhibited hyperactivity prior to dosing and wasremoved from post-dose statistical analysis. While naïve and isotypeantibody-treated subjects consistently registered an average of11,000-15,000 ambulations per day throughout the study,H4H9816P2-treated TrkB^(hu/hu) mice registered 28,260 ambulationsbetween 24-48 hours post-dosing, and registered 21,193 and 27,028ambulations from 48-72 and 72-96 hours post-dosing, respectively (Table9). H4H9816P2-treated TrkB^(hu/hu) mice registered more total ambulationcounts at each time point following antibody administration, suggestinghyperactivity to be an additional effect of H4H9816P2 injection. Incombination, these effects suggest that a single subcutaneous injectionof the TRKB agonist antibody, H4H9816P2, induced significant changes inbody weight, body composition, metabolism, and locomotion inTrkB^(hu/hu) mice.

TABLE 9 Locomotion of TrkB^(hu/hu) Mice after Dosing with TrkB AgonistAntibody H4H9816P2. Mean total Mean total Mean total Mean total Meantotal ambulations ambulations ambulations ambulations ambulations(counts) 0-24 (counts) 0-24 (counts) 24-48 (counts) 48-72 (counts) 72-96hours pre- hours post- hours post- hours post- hours post- Experimentalgroup dose (±SD) dose (±SD) dose (±SD) dose (±SD) dose (±SD) Naive (n =3) 16562 14692 14387 13279 12525 (+/−3380) (+/−2792) (+/−6126) (+/−3607)(+/−4121) Isotype Control 18105 13380 13049 11371 11468 REGN1945 (n = 4)(+/−4085) (+/−2730) (+/−3376) (+/−2552) (+/−2088) H4H9816P2 (n = 4)13292 16575 28260 21193  27028* (+/−5294) (+/−6836) (+/−19874) (+/−6668) (+/−10969)  Statistical significance determined byKruskal-Wallis One-way ANOVA with Tukey's multiple comparison post-hoctest is indicated (*= p < 0.05, **= p < 0.01, ***= p < 0.001, ****= p <0.0001, compared to isotype control group: TrkB^(hu/hu) mice dosed with50 mg/kg isotype control antibody.

Example 3. In Vivo Comparison of the Effect of TRKB Agonist AntibodyH4H9816P2 and IgG4 Isotype Control REGN1945 on TRKB Phosphorylation inthe Brain Following Stereotaxic Injection in TrkB^(hu/hu) Mice (MAID7139)

Experimental Procedure

Tyrosine receptor kinase B (TRKB) is activated through binding of itsligand brain-derived neurotrophic factor (BDNF) at the extracellularreceptor domain, which induces the dimerization and autophosphorylationof tyrosine residues in the intracellular receptor domain and subsequentactivation of cytoplasmic signaling pathways. See, e.g., Haniu et al.(1997) J. Biol. Chem. 272(40):25296-25303 and Rogalski et al. (2000) J.Biol. Chem. 275(33):25082-25088, each of which is herein incorporated byreference in its entirety for all purposes. In order to determine theeffect of a TRKB agonist antibody, H4H9816P2, on TRKB activationkinetics, a time-course study of TRKB phosphorylation following directhippocampal injection was performed in mice homozygous for a chimericmouse/human TRKB receptor in which the extracellular domain has beenhumanized (MAID 7139) (referred to as TrkB^(hu/hu) mice). TrkB^(hu/hu)mice (N=48) received bilateral stereotaxic injections of either with 2μL of either vehicle (PBS), REGN1945 hereby noted as IgG4 isotypecontrol antibody (27.5 mg/mL final concentration), or TRKB agonistantibody H4H9816P2 (27.5 mg/mL final concentration) into thehippocampus, −2 mm posterior and +1.5 mm lateral to bregma. In order tominimize tissue damage, injection and needle removal were both performedgradually over 5-minute intervals. TrkB^(hu/hu) mice were thensacrificed by CO₂ euthanasia approximately 30 minutes, 1 hour, 4 hours,or 18 hours post-injection. A terminal bleed was performed via cardiacpuncture to collect blood, and mice were then transcardially perfusedwith cold heparinized saline. The brain was carefully removed from theskull, and a 2 mm³ section of tissue surrounding the injection site wasdissected, collected in an Eppendorf tube and stored on ice. The brainsection was then lysed in 300 μL of RIPA lysis buffer (ThermoFisherScientific, Cat#89901) containing 2× protease and phosphatase inhibitors(ThermoFisher Scientific, Cat#78444) and stored on ice. The lysed tissuewas then homogenized for further processing, aliquoted and stored at−80° C.

To assess TRKB phosphorylation in the brain tissue, immuno-precipitationand western blotting was performed. Anti-human TRKB antibody H4H10108Nthat does not compete for binding with H4H9816P2 was coupled toNHS-activated Sepharose beads (prepared using manufacturer's protocol;GE Healthcare, Cat#17-0906) and washed with DPBS three times to removeany residual preservation solution. Homogenized brain lysates werethawed on ice and diluted to a concentration of 1 mg/mL (brain weight tobuffer volume) in a buffer composed of 1% NP-40, 0.1% Tween-20, proteaseand phosphatase inhibitors in TBST. The protein concentration of thehomogenized brain lysate was quantified by performing a standard BCAassay per manufacturer's instructions (Thermo Scientific Pierce,Cat#23225). For every 100 μg of protein, 15 μL of anti-human TRKBantibody (H4H10108N) NHS-activated Sepharose beads were added to thebrain lysate solution and the mixture was incubated overnight at 4° C.with gentle shaking 20 rpm (Thermo rotator). The next day, samples werecentrifuged at 1000×g for one minute, and the supernatant was thencarefully removed. Beads were subsequently washed twice with 400 μL ofTris-buffered saline (Bio-Rad, Cat#1706435) with 1% Tween-20 (SigmaAldrich, Cat#P9416) (TBST). After carefully aspirating the wash buffer,60 μL of 0.1% Trifluoroacetic acid (TFA; Sigma-Aldrich, T62200) in waterat pH 3.0 was added to each sample. The solution was mixed and allowedto stand for two minutes before being collected and transferred into aseparate tube. This process was repeated with another 60 μL of 0.1% TFAat pH 3.0. The two 0.1% TFA solutions for each sample were thencombined, and 2 μL of 1M Tris-HCl (ThermoFisher Scientific,Cat#15567-027), at pH 8.5, was added.

The solution was dried using a speed vacuum and then re-suspended andreduced with a mixture of 20 μL of 1× Laemmli Buffer (Bio-Rad,Cat#1610737) plus 355 nM 2-mercaptoethanol (BME; Gibco, Cat#21985-023).Samples were boiled at 95° C. for 10 minutes and loaded onto a 10-well,Mini-Protean 4-15% Tris-Glycine gel (Bio-Rad, Cat#4561086). Afterelectrophoresis, protein samples were transferred from the Tris-Glycinegel onto a PVDF membrane (Bio-Rad, Cat#170-4156) via the Trans-BlotTurbo Transfer System (Bio-Rad, Cat#1704156) over the course of 30minutes at a constant rate of 1.3 A and 25 V. After the transfer, themembrane was blocked with 2.5% milk (Bio-Rad, Cat#170-6406) in TBST forone hour at room temperature, and subsequently probed overnight witheither an anti-phospho-TRKB antibody (Novus, Cat#NB100-92656) diluted1:1000 in a solution of 2.5% BSA or anti-TRKB primary antibody (CellSignaling, Cat#4603) diluted to 1:1000 in 2.5% milk TBST at 4° C. on ashaker at 30 rpm. The next day, blots were washed with TBST andincubated with an anti-rabbit IgG antibody conjugated with horseradishperoxidase (Jackson, Cat#111-035-144) at 1:1000 in 1% milk in TBST for 1hour at room temperature. Blots were then washed again, developed withECL solution (PerkinElmer, Inc. Cat #RPN2106), and subsequent imageexposures were taken every 30 seconds.

Results and Conclusions

Immunoprecipitation and subsequent western blotting of protein derivedfrom TrkB^(hu/hu) mouse brain lysates demonstrated that hippocampal TRKBphosphorylation was detectable in mice injected with a TRKB agonistantibody, H4H9816P2, but not in mice treated with vehicle or isotypecontrol antibody, as shown FIG. 3. Among the time points assessed, TRKBphosphorylation peaked at 4 hours after stereotaxic injection in micedosed with H4H9816P2. TRKB phosphorylation was also detected by westernblot at 18 hours post-dosing in some, but not all mice. Conversely,injection of vehicle and IgG4 isotype control antibody did not induceTRKB phosphorylation at any time point. Western blotting also indicatedthat the total TRKB receptor levels were downregulated in some, but notall TrkB^(hu/hu) mice dosed with H4H9816P2 relative to vehicle andisotype control treated mice. Total TRKB levels appeared to be slightlydownregulated in H4H9816P2-treated subjects at 18 hours post-dosing.Thus, these results indicate that direct injection of the TRKB agonistantibody, H4H9816P2, induces phosphorylation of hippocampal TRJBreceptors in TrkB^(hu/hu) mice.

Example 4. Activation of Downstream Signaling Pathways by TrkB AgonistAntibodies in Primary Cortical Neurons from Postnatal Day 1 TrkB^(hu/hu)Mice

Experimental Procedure

All procedures were conducted in accordance with the ARVO Statement forUse of Animals in Ophthalmic and Vision Research and the RegeneronPharmaceuticals, Inc. IACUC. Primary mouse cortical neurons wereisolated and cultured from humanized TrkB mice (MAID 7139). See, e.g.,Beaudoin et al. (2012) Nat. Protoc. 7(9):1741-1754, herein incorporatedby reference in its entirety for all purposes. Western blots wereperformed to determine the effects of TrkB agonist antibodies on thedownstream pathways of Akt and Erk (p-Akt, p-Erk1/2). Primary corticalneurons from postnatal day 1 (P1) humanized TrkB mouse pups werecultured for 4 days (DIV-4) in NeuralQ Basal Medium (Global Stem, cat.#GSM-9420) supplemented with GS21 Neural Supplement (Global Stem, cat.#GSM-3100), Glutamax (Invitrogen, cat. #35050-061) andPenicillin/Streptomycin. Cells were treated with TrkB agonist antibodyH4H9816P-L1 (10 μg/mL), TrkB agonist antibody H4H9780P-L1 (10 μg/mL),TrkB agonist antibody H4H9814P-L1 (10 μg/mL), IgG4 isotype controlREGN1945 (10 μg/mL), control antibody H1M8037C-L1 (10 μg/mL), or BDNF (1μg/mL), for 15 minutes or 2 hours. Western blots were performed todetermine if the agonists have a difference in downstream signalingmaintenance and strength. Treated cells were rinsed and scraped in coldPBS containing 1% protease and phosphatase inhibitors (Sigma). Proteinconcentration was determined by Bradford protein assay (Pierce). Samples(50 μg) were separated by SDS-PAGE in 3-8% Tris-Acetate reduced gels(Novex) and transferred to a nitrocellulose membrane (Bio-Rad).

The membrane was incubated for 1 hour in blocking solution containing 5%milk and 0.1% Tween-20, pH 7.6. This was followed by overnightincubation at 4° C. in the blocking buffer containing 5% BSA, 0.1%Tween-20, and rabbit anti-phosphoTrk (Cell Signaling, cat. #9141,1:500), rabbit anti-phospho-Akt (Cell Signaling, cat. #9271, 1:1000), orrabbit anti-phospho-ERK1/2 antibody (Sigma, cat. #E7028, 1:5000).Subsequently, the labeled proteins were visualized by incubation with ahorseradish peroxidase (HRP) conjugated anti-goat, mouse or rabbit IgGfollowed by development with a chemiluminescence substrate for HRP(Pierce). To determine the amounts of total TrkB, MAPK or Akt present ineach lane, the nitrocellulose membranes were stripped of the antibodiesin stripping buffer (Pierce) for 20 min and incubated with rabbitanti-TrkB (Cell Signaling, cat. #4603, 1:1000), rabbit anti-Erk1/2 (CellSignaling, cat. #06-182, 1:1000), or rabbit anti-Akt antibody (CellSignaling, cat. #9272, 1:1000) and then visualized as described above.Beta-Actin (Sigma, cat. #A5316, 1:20000 and GAPDH (Sigma, cat. #G9295)were probed as sample loading control.

Materials

TABLE 10 mAB Clone IDs. REGN AbPID Lot H4H9816P L1 REGN1945 L1 H4H9780PL1 H4H9814P L1 H1M8037C L1 Comparator, Control antibody

TABLE 11 Reagents. Reagent/Equipment Source Identifier Lot #Penicillin/Streptomycin Invitrogen 15140 Fetal Bovine Serum Invitrogen10082-147 GS21 Neural Supplement GlobalStem GSM-3100 18130001 (50X)NeuralQ Basal Medium GlobalStem GSM-9420 18190001 Glutamax Invitrogen35050-061 Protease Inhibitor Sigma P8340 Cocktail Phosphatase InhibitorSigma P0044 034M4010V Cocktail 3 RIPA lysis buffer 1x RocklandMB-030-0250 24805 BSA Sigma A8806 Tris-Acetate 4-8% InvitrogenWG1602BX10 14022684 reduced gels BCA Protein Assay Kit Pierce 23227 ECLPierce 32209 Restore Western Blot Pierce 21059 Stripping BufferNitrocellulose membrane Bio-Rad 1620112 Laboratories

TABLE 12 Neurobasal Medium. NeuralQ Basal Medium (Global Stem, GSM-9420)50 mL GS21 Neural Supplement (50X) (Global Stem, GSM-3100) 10 mLGlutamax (Invitrogen, 35050-061) 0.5 mL Penicillin/Streptomycin 5 mL

TABLE 13 Antibodies. H4H9816P lot1 (10 μg/mL) H4H9780P lot1 (10 μg/mL)H4H9814P lot1 (10 μg/mL) REGN1945 human IgG4 lot1 (10 μg/mL) C2 H1M8037Clot1 (10 μg/mL)

TABLE 14 Western Blots. p-Trk (Cell Signaling, 9141) Rb, 1:500 totalTrkB (Cell Signaling, 4603) Rb 1:1000 p-Akt (Cell Signaling, 9271) Rb1:1000 total-Akt (Cell Signaling, 9272) Rb 1:1000 p-Erk1/2 (Sigma,E7028) 1:5000 total Erk1/2 (Cell Signaling, 06-182) Rb 1:1000 b-Actin(Sigma, A5316) Ms 1:20000 GAPDH (Sigma, G9295) HRP conjugated 1:20000Results and Conclusions

As shown in FIG. 7, while all the TrkB agonist antibodies showedactivation of the MAPK/ERK and PI3K/Akt pathways at 15 minutes after theincubation, only BDNF and H4H9814P showed TrkB phosphorylation. At 2hours after incubation, all the TrkB agonist antibodies showedactivation of TrkB.

Example 5. Pharmacokinetic Assessment of an Anti-TrkB Antibody inHumanized TrkB and Wild Type Mice

Experimental Procedure

Evaluation of the pharmacokinetics of an anti-TrkB antibody, H4H9816P2(Lot H4H9816P2-L7), was conducted in humanized TrkB (mice homozygous forchimeric mouse/human TrkB expression, TrkB^(hu/hu)) (MAID7139) and wildtype (WT) mice. Cohorts contained 5 mice per mouse strain. All micereceived a single sub-cutaneous (SC) 10 mg/kg dose. Blood samples werecollected at 6 hours and 1, 2, 3, 6, 9, 16, 21, and 30 days post-dosing.Blood was processed into serum and frozen at −80° C. until analyzed.

Circulating antibody concentrations were determined by total humanIgG4/hIgG1 antibody analysis using the GyroLab xPlore™ (Gyros, Uppsala,Sweden). Briefly, biotinylated mouse anti-human IgG4/IgG1-specificmonoclonal antibody (REGN2567; Lot RSCH15088)) diluted to 100 μg/mL inantibody dilution buffer (0.05% Tween-20+PBS) was captured on a GyrolabBioaffy 200 CD, which contained affinity columns preloaded withstreptavidin-coated beads (Dynospheres™). The standard used forcalibration in this assay was H4H9816P at concentrations ranging from0.488 to 2000 ng/mL in dilution buffer (0.5% BSA+PBS) containing 0.1%normal mouse serum (NMS). Serum samples were diluted 1:100 in theantibody dilution buffer. Human IgG captured on the anti-REGN2567-coatedaffinity columns on the CD, run at room temperature, was detected byaddition of 0.5 μg/mL Alexa-647-conjugated mouse anti-human kappamonoclonal antibody (REGN654; Lot RSCH13067) diluted in detection buffer(Rexxip F buffer); and the resultant fluorescent signal was recorded inresponse units (RU) by the GyroLab xPlore instrument. Sampleconcentrations were determined by interpolation from a standard curvethat was fit using a 5-parameter logistic curve fit using the GyrolabEvaluator Software. Average concentrations from 2 replicate experimentswere used for subsequent PK analysis.

PK parameters were determined by non-compartmental analysis (NCA) usingPhoenix®WinNonlin® software Version 6.3 (Certara, L.P., Princeton, N.J.)and an extravascular dosing model. Using the respective meanconcentration values for each antibody, all PK parameters includingobserved maximum concentration in serum (C_(max)), estimated half-lifeobserved (t_(1/2)), and area under the concentration curve versus timeup to the last measureable concentration (AUC_(last)) were determinedusing a linear trapezoidal rule with linear interpolation and uniformweighting.

Results and Conclusions

Following 10 mg/kg s.c. administration of anti-TrkB antibody, H4H9816P2,similar maximum concentrations (C_(max)) of antibody were observed byday 1 or 2 in both TrkB^(hu/hu) and WT mice (135 and 131 μg/mL,respectively). By day 9, H4H9816P2 exhibited steeper drug elimination inTrkB^(hu/hu) mice than in WT mice, indicating a target-mediated effect.Day 30 antibody concentrations were about 35-fold less in TrkB^(hu/hu)mice. Antibody exposure (AUC_(last)) for H4H9816P2 in WT mice was˜1.7-fold higher than seen in TrkB^(hu/hu) mice (1730 and 1020 d*αg/mL,respectively). WT mice also exhibited about a 3-fold increase inhalf-life (T_(1/2)) over TrkB^(hu/hu) mice (8.4 and 2.9 days,respectively).

A summary of the data for total anti-TrkB antibody concentrations aresummarized in Table 15, mean PK parameters are described in Table 16 andmean total antibody concentrations versus time are shown in FIG. 8. InFIG. 8, mice were administered a single 10 mg/kg sub-cutaneous dose onday 0. Concentrations of total H4H9816P2 in serum were measured using aGyros immunoassay. Data points on post-dose 6 hours, 1, 2, 3, 6, 9, 16,21, and 30 days indicate the mean concentration of antibody. Totalantibody concentrations of H4H9816P2 are represented as solid circlesfor TrkB^(hu/hu) mice and solid squares for wild type mice. Data areplotted as mean±SD.

TABLE 15 Mean Concentrations (±SD) of Total IgG in Serum Following aSingle 10 mg/kg Sub-Cutaneous Injection of H4H9816P2 in TrkB^(hu/hu) andWild Type Mice over Time. Total mAb Concentration in Mouse Serum 10mg/kg Time Mean Antibody (d) (μg/mL) +/−SD TrkB^(hu/hu) Mice 0.25 72.424.06 1 132.0 18.0 2 124.9 15.9 3 113.4 11.8 6 78.72 9.98 9 37.74 14.0 165.592 4.97 21 2.060 2.11 30 0.447 0.506 WT Mice 0.25 56.73 14.5 1 120.86.26 2 131.2 7.54 3 125.7 7.46 6 101.9 11.4 9 75.94 7.06 16 42.61 16.121 27.75 16.9 30 15.52 13.0 Abbreviations: Time = time in days postsingle-dose injection; d = day of study; SD = standard deviation.

TABLE 16 Summary of Pharmacokinetic Parameters. H4H9816P2 ParameterUnits TrkB^(hu/hu) Mice WT Mice C_(max) μg/mL 135 ± 15  131 ± 7.5T_(1/2) d 2.94 ± 1.1 8.36 ± 3.9 AUC_(last) d · μg/mL 1020 ± 150 1730 ±310 PK parameters were derived from mean concentration versus timeprofiles. T_(1/2) and AUC_(last) are based on concentrations out to day30. Abbreviations: C_(max) = peak concentration; AUC = area under theconcentration-time curve; AUC_(last) = AUC computed from time zero tothe time of the last positive concentration; T_(1/2) = terminalhalf-life of elimination.

Example 6. Generation of Rats Comprising a Humanized TRKB Locus

A large targeting vector comprising a 5′ homology arm comprising 7 kb ofthe rat TrkB locus and 3′ homology arm comprising 47 kb of the rat TrkBlocus was generated to replace a region of 68.5 kb from the rat TrkBgene encoding the rat TRKB extracellular domain with 74.4 kb of thecorresponding human sequence of TRKB. Generation and use of largetargeting vectors (LTVECs) derived from bacterial artificial chromosome(BAC) DNA through bacterial homologous recombination (BHR) reactionsusing VELOCIGENE® genetic engineering technology is described, e.g., inU.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotechnol.21(6):652-659, each of which is herein incorporated by reference in itsentirety for all purposes. Generation of LTVECs through in vitroassembly methods is described, e.g., in US 2015/0376628 and WO2015/200334, each of which is herein incorporated by reference in itsentirety for all purposes. Information on rat and human TRKB is providedin Table 17. A description of the generation of the large targetingvector is provided in Table 18.

TABLE 17 Rat and Human TRKB/NTRK2. Official NCBI Primary Genomic SymbolGeneID Source RefSeq mRNA ID UniProt ID Assembly Location Rat Ntrk225054 RGD: 3213 NM_012731.2 Q63604 RGSC 5.0/rn5 Chr 17: 8,156,432-8,464,507 (−) Human Ntrk2 4915 HGNC: 8032 AF410899 Q16620 GRCh38/hg38Chr 9: 84,669,778- 85,027,070 (+)

TABLE 18 Rat TrkB/Ntrk2 Large Targeting Vector. Genome Build Start EndLength (bp) 5′ Rat Arm RGSC 5.0/rn5 Chr17: 8,470,615 Chr17: 8,463,3797,236 Human Insert GRCh38/hg38 Chr9: 84,670,730 Chr9: 84,745,139 74,4093′ Rat Arm RGSC 5.0/rn5 Chr17: 8,394,967 Chr17: 8,347,889 47,078

Specifically, a region starting in exon 2 (coding exon 1; from aminoacid 32, preserving signal peptide) through exon 10, including the first50 base pairs of intron 10 and all introns between exons 2 and 10 (i.e.,between coding exon 1 and exon 10) was deleted from the rat TrkB locus(preserving the rat transmembrane domain encoded by exons 10 and 11). Aregion including exon 2/coding exon 1 (from amino acid 32, beginningafter the signal peptide) through exon 10, including the first 66 basepairs of intron 10 and all introns between exons 2 and 10 (i.e., betweencoding exon 1 and exon 10) was inserted in place of the deleted ratregion (preserving the rat transmembrane domain encoded by exons 10 and11).

Sequences for the rat TRKB signal peptide, extracellular domain,transmembrane domain, and cytoplasmic domain are set forth in SEQ IDNOS: 55-58, respectively, with the corresponding coding sequence setforth in SEQ ID NOS: 67-70, respectively. Sequences for the human TRKBsignal peptide, extracellular domain, transmembrane domain, andcytoplasmic domain are set forth in SEQ ID NOS: 59-62, respectively,with the corresponding coding sequence set forth in SEQ ID NOS: 71-74,respectively. The expected encoded chimeric TRKB protein is has rat TRKBtransmembrane and intracellular domains, a rat TRKB signal peptide, anda human TRKB extracellular domain. See FIG. 4. An alignment of the ratand human TRKB proteins in FIG. 6. The rat and human TrkB/TRKB codingsequences are set forth in SEQ ID NOS: 10 and 11, respectively. The ratand human TRKB protein sequences are set forth in SEQ ID NOS: 2 and 3,respectively. The sequences for the expected chimeric rat/human TRKBcoding sequence and the expected chimeric rat/human TRKB protein are setforth in SEQ ID NOS: 13 and 5, respectively.

To generate the mutant allele, CRISPR/Cas9 components including fourguide RNAs (guide RNA target sequences set forth in SEQ ID NOS: 41-44)were introduced into rat embryonic stem cells together with the largetargeting vector. Specifically, 4×10⁶ rat ES cells (Dark Agouti lineDA2B) were electroporated with the following: 2 mg TrkB LTVEC; 5 mg Cas9expression plasmid; and 5 mg each of the gRNAs: gU, gU2, gD and gD2. Theelectroporation conditions were: 400 V voltage; 100 mF capacitance; and0 W resistance. Antibiotic selection was performed using G418 at aconcentration of 75 mg/mL. Colonies were picked, expanded, and screenedby TAQMAN®. See FIG. 5. Loss-of-allele assays were performed to detectloss of the endogenous rat allele, gain-of-allele assays were performedto detect gain of the humanized allele, and CRISPR and retention assayswere performed using the primers and probes set forth in Table 19.

TABLE 19 Screening Assays. Primer/ Assay Description Probe SequencernoTU Upstream Fwd GGGCTCAGGCAGGTATATGTTG (SEQ ID NO: 26) LOAProbe (FAM) ACAGATGCTGTCCCAAACATAGCAAGA (SEQ ID NO: 27) RevCCAACCCTAAGCCAGTGAAACAG (SEQ ID NO: 28) rnoTM Middle FwdGCAGACACTGGATGGGTCA (SEQ ID NO: 32) LOA Probe (FAM)CCATTCGCGAGTTATGAGAAGCTGCA (SEQ ID NO: 33) RevACAGGGTTAGCTGGTGAATGGA (SEQ ID NO: 34) rnoTD Downstream FwdGTGCTGGAGACCAGGAGACT (SEQ ID NO: 29) LOA Probe (Cal-TGCCATACTCAGTTTATACGGTGCTGAC (SEQ ID NO: 30) Orange) RevGCCTGGTGGCTCAGTCAATG (SEQ ID NO: 31) 7138 hU Upstream FwdAGGTGGGTAGGTCCTGGAAGTG (SEQ ID NO: 14) Human Probe (FAM)AATGCTGTCCCAAGAGTGGG (SEQ ID NO: 15) Insertion RevGTCCTGCATCCCTTGTCTTTG (SEQ ID NO: 16) 7138 hD Downstream FwdATGTGGGCGTTGTGCAGTCTC (SEQ ID NO: 17) Human Probe (Cal)CGCTGCAGTGCATTGAACTCAGCA (SEQ ID NO: 18) Insertion RevCTGTGGAGGGACGTGACCAG (SEQ ID NO: 19) rnoTAU2 Upstream FwdTCGGAGCACAGGACTACAG (SEQ ID NO: 35) Retention Probe (FAM)CAAGAGGAACTGTGTCCAGGAAAGC (SEQ ID NO: 36) RevAGCGTGCCTCACCTAACCTCTA (SEQ ID NO: 37) rnoTAD2 Downstream FwdGCACAGCACTGTAAAGGCA (SEQ ID NO: 38) Retention Probe (Cal)ACGGAACTCGAAGGAATTGGTATTGTTGT (SEQ ID NO: 39) RevACACAGCTATGGGAGAAAGACTG (SEQ ID NO: 40) rnoTGU Upstream FwdCTGGGTGATTGGGACTGAGAAAG (SEQ ID NO: 45) CRISPR Probe (FAM)CAGCCTTGAAAGTATGGCTTGGGC (SEQ ID NO: 46) Assay RevGCACTCGCCAACCGGAAG (SEQ ID NO: 47) rnoTGD Downstream FwdGACCAGCTCACCCTTACTTATGG (SEQ ID NO: 48) CRISPR Probe (Cal)ACTGAATGCCAAGGGTGCGTTGA (SEQ ID NO: 49) Assay RevTCTTGGAAATCCGCTGAAGAGTT (SEQ ID NO: 50)

Modification-of-allele (MOA) assays including loss-of-allele (LOA) andgain-of-allele (GOA) assays are described, for example, in US2014/0178879; US 2016/0145646; WO 2016/081923; and Frendewey et al.(2010) Methods Enzymol. 476:295-307, each of which is hereinincorporated by reference in its entirety for all purposes. Theloss-of-allele (LOA) assay inverts the conventional screening logic andquantifies the number of copies in a genomic DNA sample of the nativelocus to which the mutation was directed. In a correctly targetedheterozygous cell clone, the LOA assay detects one of the two nativealleles (for genes not on the X or Y chromosome), the other allele beingdisrupted by the targeted modification. The same principle can beapplied in reverse as a gain-of-allele (GOA) assay to quantify the copynumber of the inserted targeting vector in a genomic DNA sample.

Retention assays are described in US 2016/0145646 and WO 2016/081923,each of which is herein incorporated by reference in its entirety forall purposes. Retention assays distinguish between correct targetedinsertions of a nucleic acid insert into a target genomic locus fromrandom transgenic insertions of the nucleic acid insert into genomiclocations outside of the target genomic locus by assessing copy numbersof DNA templates from 5′ and 3′ target sequences corresponding to the 5′and 3′ homology arms of the targeting vector, respectively.Specifically, retention assays determine copy numbers in a genomic DNAsample of a 5′ target sequence DNA template intended to be retained inthe modified target genomic locus and/or the 3′ target sequence DNAtemplate intended to be retained in the modified target genomic locus.In diploid cells, correctly targeted clones will retain a copy number oftwo. Copy numbers greater than two generally indicate transgenicintegration of the targeting vector randomly outside of the targetgenomic locus rather than at the target genomic locus. Copy numbers ofless than generally indicate large deletions extending beyond the regiontargeted for deletion.

CRISPR assays are TAQMAN® assays designed to cover the region that isdisrupted by the CRISPR gRNAs. When a CRISPR gRNA cuts and creates anindel (insertion or deletion), the TAQMAN® assay will fail to amplifyand thus reports CRISPR cleavage.

The positive clone CB1 was thawed, expanded, and reconfirmed by TAQMAN®.CB1 was also confirmed by successful PCR from the 5′ end of the humanreplacement sequence to the flanking rat genomic sequence, beyond theend of the 5′ homology arm. The PCR amplicon was confirmed as correct bysequencing of the ends.

F0 and F1 rats were generated using methods as described in US2014/0235933, US 2014/0310828, WO 2014/130706, and WO 2014/172489, eachof which is herein incorporated by reference in its entirety for allpurposes. In such methods, confirmed targeted rat ES cell clones (e.g.,Dark Agouti ES cell clones) are microinjected into blastocysts (e.g.,Sprague Dawley (SD) blastocysts), which are then transferred topseudopregnant recipient females (e.g., SD recipient females) forgestation using standard techniques. Chimeras are identified (e.g., bycoat color), and male F0 chimeras are bred to female wild-type rats ofthe same strain (e.g., SD females). Germline (e.g., agouti) F1 pups arethen genotyped for the presence of the targeted allele. All experimentsperformed in humanized TRKB rats as described below were performed inrats in which the self-deleting selection cassette was self-deleted.

Example 7. In Vivo Comparison of the Effect of TRKB Agonist AntibodyH4H9816P2 and IgG4 Isotype Control REGN1945 on Retinal Ganglion Cell(RGC) Survival TrkB^(hu/hu) Rats

Experimental Procedure

All procedures were conducted in accordance with the ARVO Statement forUse of Animals in Ophthalmic and Vision Research and the RegeneronPharmaceutical Inc. IACUC. Adult female TrkB humanized rats(MAID100010), 8-10 weeks old, each weighing 200-250 g, were used. Allsurgical procedures on rats were performed under general anesthesiausing an intraperitoneal injection of ketamine (63 mg/kg) and xylazine(6.0 mg/kg). Eye ointment containing erythromycin (0.5%, Bausch & Lomb)was applied to protect the cornea.

Intraorbital Optic Nerve Axotomy and Intravitreal Injection.

The left optic nerve (ON) was exposed intraorbitally, its dura wasopened. ON was transected about 1.5 mm behind the globe. Care was takento avoid damaging the blood supply to the retina. Intravitrealinjections were performed just posterior to the pars plana with a pulledglass pipette connected to a 50 μL Hamilton syringe. Care was taken notto damage the lens. Rats with any significant postoperativecomplications (e.g., retinal ischemia, cataract) were excluded fromfurther analysis. Animals were allocated to different experimentalgroups. One control group received intravitreal injections of 3 μLisotype control REGN1945 (46.6 μg/μL); the other group receivedinjection of 3 μL anti-human TRKB antibody H4H9816P2 (45.7 μg/μL) at 3and 10 days after ON axotomy.

Immunohistochemical Staining and Counting of Viable Retinal GanglionCells (RGCs).

BRN3A (brain-specific homeobox/POU domain protein 3A) was used as amarker for surviving retinal ganglion cells (RGCs), because it has beenshown to be an efficient and reliable method for selective labelling ofviable RGCs in retinal whole mounts after ON injury. See, e.g.,Nadal-Nicolás et al. (2009) Invest. Ophthalmol. Vis. Sic.50(8):3860-3868, herein incorporated by reference in its entirety forall purposes. To immunostain for BRN3A, retinas were blocked in 10%normal donkey serum and 0.5% Triton X-100 for 1 hr, then incubated inthe same medium with BRN3A antibody (1:400; Cat#: sc-31984, Santa Cruz)2 hr at room temperature. After further washes retinas were incubatedwith Alexa594-conjugated donkey anti-goat secondary antibody (1:400;Cat#: A-11058, Invitrogen) overnight at 4° C.

Results and Conclusions

To assess the effect of the TRKB agonist antibody on RGC survival invivo, we used a complete optic nerve transection model. TRKB agonistantibody (H4H9816P2) or isotype control antibody was applied at 3 and 10days after surgery. Animals were euthanized 14 days after axotomy. TheRGC density in the uninjured contralateral eye is similar in the threeTRKB genotypes (homozygous humanized, heterozygous humanized, and wildtype), averaging around 1600 per mm² as shown in Table 20. The densityof surviving RGCs was assessed in retinal whole mounts using BRN3Astaining. We found that in homozygous TrkB^(hu/hu) humanized rats, TRKBagonist antibody (H4H9816P2) significantly (p<0.01, Mann-Whitney test)increased RGC survival compared with controls (685±106 vs. 255±66 RGCsper mm²). In heterozygous TrkB^(hu/+) humanized rats, there is alsosignificant (p<0.05, Mann-Whitney test) survival effect of TrkB agonistAb (444±90 vs. 208±50 RGCs per mm²). In wild type rats, there was aslight but not significant increase of RGC number in rats treated withTRKB agonist antibody compared to isotype control (Table 21). Inconclusion, the TRKB agonist antibody (H4H9816P2) significantlyincreased RGC survival in TrkB^(hu/hu) humanized rats.

TABLE 20 RGC Quantification (RGCs/mm²) in Uninjured Control Eye. hu/huhu/+ +/+ 1637.3 1720.4 1636.3 1551.5 2064.6 1670.2 1651.4 1738.8 1873.41628.2 2029.8 1725.4 1804.7 1929.6 1973.4 1741.3 1645.9 1739.7 1761.51698.8 1787.5 1862.5 1914.0 1779.4

TABLE 21 RGC Quantification (RGCs/mm²) after Optic Nerve Injury.H4H9816P2 Isotype control Ab A: Y1 A: Y2 A: Y3 A: Y4 A: Y5 B: Y1 B: Y2B: Y3 B: Y4 B: Y5 Hu/Hu 790.1 737.1 756.3 587.8 555.7 322.8 295.0 286.9171.3 197.9 Hu/+ 530.4 457.5 522.9 390.6 319.2 231.0 184.6 265.1 151.3+/+ 320.9 355.5 256.9 342.7 112.3

Example 8. Heavy and Light Chain Variable Region Amino Acid Sequences ofAnti-TRKB Antibodies Used in Examples

Several fully human anti-TRKB antibodies (i.e., antibodies possessinghuman variable domains and human constant domains) were tested in theexamples, including those designated as H4H9780P, H4H9814P, andH4H9816P2. Table 22 sets forth the amino acid sequence identifiers ofthe heavy and light chain variable regions and CDRs of selectedanti-TRKB antibodies used in the examples. Table 23 sets forth thenucleic acid sequence identifiers of the heavy and light chain variableregions and CDRs of selected anti-TRKB antibodies used in the examples.These antibodies are described in more detail in U.S. patent applicationSer. No. 16/202,881, filed Nov. 28, 2018, which is herein incorporatedby reference in its entirety for all purposes.

TABLE 22 Amino Acid SEQ ID NOS for Anti-TRKB Antibodies. Ab Name VHHCDR1 HCDR2 HCDR3 VK LCDR1 LCDR2 LCDR3 H4H9780P 79 81 83 85 87 89 91 93H4H9814P 95 97 99 101 103 105 107 109 H4H9816P2 111 113 115 117 119 121123 125

TABLE 23 Nucleic Acid SEQ ID NOS for Anti-TRKB Antibodies. Ab Name VHHCDR1 HCDR2 HCDR3 VK LCDR1 LCDR2 LCDR3 H4H9780P 78 80 82 84 86 88 90 92H4H9814P 94 96 98 100 102 104 106 108 H4H9816P2 110 112 114 116 118 120122 124

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g., “H4H”), followed by a numericalidentifier (e.g., “9780,” “9816,” etc., as shown in Table 22), followedby a “P” or “P2” suffix. The H4H prefix in the antibody designationsindicates the particular Fc region isotype of the antibody. Thus,according to this nomenclature, an antibody may be referred to hereinas, e.g., “H4H9780P,” which indicates a human IgG4 Fc region. Variableregions are fully human if denoted by the first “H” in the antibodydesignation. As will be appreciated by a person of ordinary skill in theart, an antibody having a particular Fc isotype can be converted to anantibody with a different Fc isotype (e.g., an antibody with a mouseIgG1 Fc can be converted to an antibody with a human IgG4, etc.), but inany event, the variable domains (including the CDRs)—which are indicatedby the numerical identifiers shown in Table 22—will remain the same, andthe binding properties to antigen are expected to be identical orsubstantially similar regardless of the nature of the Fc domain.

Example 9. Neuroprotective Effect of Anti-Human TrkB Agonist Antibodiesin Humanized TrkB Rats

The experiments below were undertaken to evaluate the neuroprotectiveeffect of the endogenous TRKB agonist, brain-derived neurotrophic factor(BDNF), and a TRKB agonist monoclonal antibody (mAb) in wild-type (WT)mice and rats and in humanized TrkB mice and rats.

The in vitro effects of BDNF and TRKB Ab were quantified by cellsurvival assays using differentiated human neuroblastoma cell lineSH-SY5Y. In vitro, BDNF or TRKB Ab significantly increased cell survivalin retinoic-acid-differentiated SH-SY5Y cells. The effects showed bellshaped dose responses with the optimal dose of 1 μg/mL for BDNF or 10μg/mL for TRKB Ab. Neuroblastoma cell line SH-SY5Y was cultured indifferentiation media containing all-trans 10 μM retinoic acid for 4days. The culture was changed to serum-free differentiation mediacontaining different dose of antibodies (0.01-100 μg/mL). Two dayslater, CCK8 reagent was added, plates were incubated for 3-4 hours, andOD450 was measured to determine percentage of surviving cells. Data werenormalized to the serum-free media without antibodies. As shown in FIG.9, TRKB mAbs (TrkB mAb1 is H4H9816P2; TrkB mAb2 is a control TrkBagonist antibody) dose-dependently increased the survival of SH-SY5Ycells. Human isotype control had no effect on SH-SY5Y cell survival.Serum-free media without antibodies resulted in 100% survival.

Retinas from P2 C57BL/6J mice were then dissected and dissociated.Retinal ganglion cells were purified by immuno-panning and cultured in a96-well plate with treatment or no treatment. After 24 hours, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was addedto each well to calculate cell survival for each group. As shown in FIG.10, BDNF had a bell-shaped response curve and optimal dose at 1 μg/mL.TrkB mAb2 (a control TrkB agonist antibody with affinity for human TrkB,mouse TrkB, and rat TrkB) may have a bell-shaped curve as well at higherdoses but shows neuroprotective effect.

To test the in vivo neuroprotective effect, WT and humanized TrkB miceand rats were used. Animals received intravitreal (IVT) injections ofBDNF or TRKB mAb day 3 and 10 post-optic-nerve transection (ONT).Retinal ganglion cell (RGC) number was quantified using HALO software(Indica Labs) at 1 week for mouse or 2 weeks for rat after optic nervetransection by Brn3a IHC on retinal flat mounts.

RGC death in TrkB^(hu/hu) mice was similar to WT mice at 1 or 2 weeksafter optic nerve transection. BDNF or TRKB Ab had small or nosignificant neuroprotective effect in WT or TrkB^(hu/hu) mice. Incontrast, there was significant RGC neuroprotection in TrkB^(hu/hu) ratswith IVT TRKB Ab. A decrease in body weight was observed in TrkB^(hu/hu)mice but not rats after IVT TRKB Ab treatment. BDNF had no effect onbody weight in either mouse or rat.

FIGS. 11A and 11B show the results of an experiment assessingneuroprotection in an optic nerve transection model in WT mice and rats.In FIG. 11A, 8-9 week old Dark Agouti rats were given BDNF (5 μg), TrkBmAb2 (18 μg), isotype control antibody (18 μg), or vehicle controlintravitreally at 3 days and 10 days after transection. TrkB mAb2 is acontrol TrkB agonist antibody with affinity for human TrkB, mouse TrkB,and rat TrkB. Retinas were dissected and stained for retinal ganglionicells 14 days after transection. BDNF and TRKB mAb showed significantneuroprotection as measured by retinal ganglion cell (RGC) density. InFIG. 11B, 8-week-old C57BL/6J WT mice were given BDNF (2.5 m), TrkB mAb2(10 m), isotype control antibody (10 m), or vehicle controlintravitreally at 3 days and 10 days after transection. TrkB mAb2 is acontrol TrkB agonist antibody with affinity for human TrkB, mouse TrkB,and rat TrkB. There was no significant neuroprotection. Thus, BDNF andTRKB mAb treatment resulted in significant increases in RGC density indissected retinas in wild type rats after optic nerve transection,whereas no significant effect on RGC density was observed in the samemodel in wild type mice.

FIGS. 12A and 12B show BDNF dose response in WT mice and rats. In FIG.12A, BDNF dose response in an optic nerve crush (ONC) model in WT miceshows a small window of neuroprotection. FIG. 12B shows a BDNF doseresponse in an optic nerve transection model in WT rat from 0.13 μg to30 μg. There is bell-shaped response similar to the in vitro data, withthe optimal dose at 0.8 μg. Retinas were dissected and stained forretinal ganglioni cells 14 days after transection. Thus, BDNF treatmentresulted in much more pronounced dose response curve as measured by RGCdensity in dissected retinas in wild type rats after optic nervetransection compared to the much less pronounced BDNF dose responsecurve in as measured by RGC density in dissected retinas in wild typemice after optic nerve crush.

Neuroprotective effect of TRKB Abs was next tested in humanized TrkBrats. The results in FIGS. 13A and 13B show that intravitreal injectionof TRKB mAb in optic-nerve-transected humanized TrkB rats showsignificant neuroprotection of retinal ganglion cells. Human TRKBhomozygous, human TRKB heterozygous, or wild-type TrkB rats were giveneither TrkB mAb1 or isotype control antibody intravitreally (3 μL) at 3and 10 days after optic nerve transection. Fourteen days aftertransection, retinas were dissected and stained for RGCs. The rats werefemales that were 17-19 weeks old. As shown in FIG. 13A, rats treatedwith TrkB mAb1 (H4H9816P2) showed neuroprotection in all three genotypescompared to corresponding rats treated with isotype control antibody.Isotype-control-treated homozygous and heterozygous rats for human TRKBhave higher RGC density than isotype-control-treated wild-type rats.FIG. 13B shows no RGC number difference in the naïve eyes betweengenotypes. FIG. 13C shows body weight of human TRKB homozygous micegiven either TrkB agonist antibody (H4H9816P2) or isotype controlantibody (REGN1945) at 14 days after transection.

Rat retinal whole-mount RGC isodensity maps were then created showingBrn3a labeled cells of non-injured and treated injured eyes in the threegenotypes (data not shown). Whole mount reconstruction was prepared withthe aid of motorized stage on fluorescence microscope (Nikon EclipseTi). RGCs were counted using an image analysis software (HALO®; IndicaLabs, Corrales, N. Mex., USA). Isodensity maps were generated throughMatlab. Higher RGC density was observed with the humanized TrkB ratstreated with TrkB mAb1 (H4H9816P2) compared to theisotype-control-treated rats (data not shown).

Taken together, the data shown in FIGS. 11A, 11B, 12A, 12B, and 13A-13Cdemonstrate that intravitreal administration of TRKB agonist mAb has asignificant neuroprotective effect after optic nerve injury in humanizedTrkB rats, in contrast to the small or no significant neuroprotectiveeffect observed after optic nerve injury in humanized TrkB mice.

To further evaluate the effect of TRKB agonist antibodies on RGCsurvival in rats in the optic-nerve transection (ONT) model, adose-response study was undertaken. Human TRKB homozygous rats(MAID100010; 75% SD, 25% DA) that were 1-9 months old were used. Sixrats were used in each group. Human TRKB homozygous rats were givendifferent doses of either TrkB mAb1 or isotype control antibody(REGN1945) intravitreally (3 μL) at 3 and 10 days after optic nervetransection. Fourteen days after transection, retinas were dissected andstained for RGCs. As shown in FIG. 14, TrkB mAb1 dose-dependentlyincreased RGC survival in the TrkB humanized rats.

Next, the neuroprotective effect of different TrkB agonist antibodieswas compared in human TRKB homozygous rats in the optic-nervetransection (ONT) model. Humanized TrkB rats (MAID100010; 75% SD, 25%DA) that were 8-10 weeks old were used. Five to six rats were used ineach group. Human TRKB homozygous rats were given either H4H9816P2-L9(10 μg), H4H9814P-L9 (10 μg), H4H9780P-L5 (10 μg), a combination of allthree (3.3 μg each), or isotype control antibody (REGN1945; 10 μg)intravitreally (3 μL) at 3 and 10 days after optic nerve transection.Fourteen days after transection, retinas were dissected and stained forRGCs. The results are shown in FIGS. 15A and 15B. Each TrkB agonistantibody had a neuroprotective effect compared to the isotype controlantibody. Body weight in each group was similar (data not shown).

In contrast, the TrkB agonist antibodies H4H9780P and H4H9814P did nothave any neuroprotective effect in wild type rats. Neuroprotectiveeffect was assessed in wild type rats using the optic-nerve transection(ONT) model. Female wild type rats that were 8-10 weeks old were used.Five to six rats were used in each group. Wild type rats were giveneither H4H9780P (120 μg), H4H9814P (120 μg), or isotype control antibody(REGN1945; 120 μg) intravitreally (3 μL) at 3 and 10 days after opticnerve transection. Fourteen days after transection, retinas weredissected and stained for RGCs. As shown in FIG. 16, neither TrkBagonist antibody had a significant neuroprotective effect in wild typerats.

In addition, TrkB agonist antibody (H4H9780P) did not have aneuroprotective effect in human TRKB homozygous mice. Male human TRKBhomozygous mice (MAID7139; 75% C57BL/6, 25% 129) that were 5 months oldwere used. Five to six mice were used in each group. Human TRKBhomozygous mice were given either H4H9780P (40 μg per eye) or isotypecontrol antibody (REGN1945; 40 μg per eye) intravitreally (1 μL) at 3and 10 days after optic nerve transection. Fourteen days aftertransection, retinas were dissected and stained for RGCs. As shown inFIGS. 17A and 17B, the TrkB agonist antibody did not have aneuroprotective effect in human TRKB homozygous mice in contrast to theneuroprotective effect seen in human TRKB homozygous rats. FIG. 17Cshows body weight of human TRKB homozygous mice given either H4H9780P orisotype control antibody at 14 days after transection.

We claim:
 1. A genetically modified rat whose genome comprises agenetically modified endogenous rat TrkB locus comprising a humanizedTrkB gene comprising a replacement of an endogenous rat TrkB genomicsequence encoding a rat tropomyosin receptor kinase B (TRKB) proteinextracellular domain with a corresponding human TRKB genomic sequenceencoding a human TRKB protein extracellular domain, wherein theendogenous rat TrkB genomic sequence comprises a region starting fromthe codon in exon 2 encoding amino acid 32 through exon 10, includingall introns between exons 2 and 10, wherein the corresponding human TRKBgenomic sequence comprises a region starting from the codon in exon 2encoding amino acid 32 through exon 10, including all introns betweenexons 2 and 10, wherein the humanized TrkB gene encodes: a humanizedTRKB protein comprising: an endogenous rat TRKB protein signal peptide;the human TRKB protein extracellular domain; an endogenous rat TRKBprotein transmembrane domain; and an endogenous rat TRKB proteincytoplasmic domain, wherein the humanized TRKB protein comprises thesequence set forth in SEQ ID NO: 5, wherein the humanized endogenousTrkB gene is under the control of an endogenous rat TrkB promoter at thegenetically modified endogenous rat TrkB locus, wherein the ratexpresses the humanized TRKB protein, wherein the rat is homozygous forthe genetically modified endogenous rat TrkB locus, and whereintreatment of the rat with a human TRKB agonist antibody results in aneuroprotective effect on retinal ganglion cell viability after opticnerve injury.
 2. The genetically modified rat of claim 1, wherein thehumanized TRKB protein is encoded by the nucleotide sequence set forthin SEQ ID NO:
 13. 3. A method of assessing activity of ahuman-TRKB-targeting reagent in vivo, comprising: (a) administering thehuman-TRKB-targeting reagent to the genetically modified rat of claim 1;and (b) assessing the activity of the human-TRKB-targeting reagent inthe genetically modified rat of step (a) as compared to an untreatedcontrol rat.
 4. An isolated rat cell of the genetically modified rat ofclaim 1, wherein the rat cell's genome comprises the geneticallymodified endogenous rat TrkB locus comprising the humanized TrkB genecomprising the replacement of the endogenous rat TrkB genomic sequenceencoding the rat TRKB protein extracellular domain with thecorresponding human TRKB genomic sequence encoding the human TRKBprotein extracellular domain, wherein the cell expresses the humanizedTRKB protein.
 5. The genetically modified rat of claim 1, wherein thehuman TRKB protein extracellular domain is encoded by the nucleotidesequence set forth in SEQ ID NO:
 72. 6. The genetically modified rat ofclaim 1, wherein the endogenous rat TRKB protein signal peptide isencoded by the nucleotide sequence set forth in SEQ ID NO:
 67. 7. Thegenetically modified rat of claim 1, wherein the endogenous rat TRKBprotein transmembrane domain is encoded by the nucleotide sequence setforth in SEQ ID NO:
 69. 8. The genetically modified rat of claim 1,wherein the endogenous rat TRKB protein cytoplasmic domain is encoded bythe nucleotide sequence set forth in SEQ ID NO:
 70. 9. The geneticallymodified rat of claim 1, wherein the endogenous rat TRKB protein signalpeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:67, the endogenous rat TRKB protein transmembrane domain is encoded bythe nucleotide sequence set forth in SEQ ID NO: 69, and the endogenousrat TRKB protein cytoplasmic domain is encoded by the nucleotidesequence set forth in SEQ ID NO: 70.